IGEM:Paris Bettencourt 2012/Notebook/RE group/Lab notes

From OpenWetWare

Jump to: navigation, search

Contents

28.08.2012

Sequencing results

24.08.2012

Run a gel

Gel purify

Prepare for sequencing

Digestions

23.08.2012

Digestions

Run a Gel

Miniprep

Run gel of the digestions of this morning

gel purify

ligation results

colony PCR

Liquid cultures

22.08.2012

Digestion of the gblock

Column purify

Ligation

Gel

Transformation

Miniprep liquid cultures & glycerol stock

Results of ligation

Colony PCR

Liquid cultures

21.08.2012

Ligation results and results of the K175027 transformation

  • pBAD+pSB3C5 (pRG115): a few colonies in the 2uL plate (almost none in the neg control plate); many colonies in the 8uL plate (very few in the neg control plate)
  • pRha+pSB3C5 (pRG116): lots of colonies in the 2uL plate (a few in the neg control plate); lots +++ colonies in the 8uL plate (quite a lot in the neg control plate)
  • K175027 transformation: 1 colony in the 0,5uL plate; 0 colonies in the 2uL plate (it seems that the transformation was unsuccessful but we will still PCR and liquid culture the colony we got).

Colony PCR

  • 3 colonies per plate
  • 12 reactions from ligaton plate + 1 from K175027 transformation plate + 1neg control = 14 reactions.

Liquid cultures

  • We pick 3 colonies per plate (same then the culture PCR)
  • 13 tubes LB+Cm and 1 tube LB+Amp

Run a gel

  • expected sizes:
  • pRG115: 286bp (actually 411bp! I don't know why but I made a mistake in the expected size, so I thought the gel results were wrong for this construct)
  • pRG116: 402bp
  • K175027: 268bp

Details:

  • 2%agarose, 100bp ladder

Results:

  • pRG115: not ok (but because of an error on my some, some were ok actually: wells 1 and 5)
  • pRG116: ok wells 9, 12, 13
  • K175027: ok (so although the transformation seemed unsuccessful at first since there was only one colony, this colony is satisfactory)

Gel purification (gel runed 20.08.12)

  • pRG106: 5,6ng/uL
  • pRG109: 8,4 ng/uL
  • pRG112: 7,4 ng/uL
  • K175027: 6,7ng/uL

Ligation

  • pRG106 + K175027
  • pRG109 + K175027
  • pRG112 + K175027

A standard protocol was used

Transformation

  • A standard protocol was used.
  • We transformed 2uL and 8uL for each reaction
  • LBA+Cm plates

20.08.2012

Ligations

  1. pRHA (X,P) + pSB3C5 (X, P)
  2. pRG004 (S, E) + pSB3C5
  • 10uL reactions: 1uL of fermentas T4 ligase buffer + 0,5uL of fermentas T4 ligase enzyme + ration 1:5 vector/insert + H20 up to 10uL
  • Flick the tube gently to mix
  • Incubate at 22°c for 1h

Transformation of ligation products

  • standard protocol was used
  • 2uL and 8uL of digestion product was transformed
  • 4 plates + 4 neg control = 8 plates (LBA + Cm)
  • We also transformed K175027: 0.5ul and 2uL

Digestions

  • pRG106: E & X
  • pRG109: E & X
  • pRG112: E & X
  • K175027: E & S

Stadard protocol was used (incubation for 1h at 37°C)

Gel

Expected size of parts:

  • pRG106: 3746
  • pRG109: 3671
  • pRG112: 3594
  • K175027: 73

Details:

  • 2% agarose gel, 100bp ladder:pRG112, K175027
  • 1% agarose gel, 1kb ladder: pRG 106, pRG109

NB: The USB key did not save all the images so the two I put here are not great (one is one of the gel already cut. The other one is before I put the other gel back so I can migrate it a little longer)

17.08.2012

Digestions

  • pSB3C5: X & P
  • pRHA: X & P
  • pRG 105(pSB3C5+RBS+GFP): E & X
  • pRG006(pLac): E & S
  • pRG004(pBAD): E & S
  1. 40uL reactions: 4uL of 10*fast digest buffer, 2uL of each enzyme, 1pg of DNA and H20 to 40uL.
  2. Mix gently and spin down
  3. incubate at 37°c for 1h

Gel

Expected size of pieces:

  • pSB3C5: 3828bp
  • pRG105: 3605bp
  • pRG006: 55bp
  • pRG004: 130bp

Details:

  • 2% agarose gel, 100bp ladder: pRG006, pRG 004
  • 1% agarose gel, 1kb ladder: pRG105, pSB3C5

Gel purification

  • pSB3C5 (X & P):8,3 ng/uL
  • pRG105 (X & E): 11,4 ng/uL
  • pRG004 (S & E): 89,8ng/uL

NB: we do not gel purify pRG006 (pLAC) because it happens that SG group just made the construct pLac+pSB3C5, so we don't need to make it.

Column purify pRHA

  • pRHA (X & P): 26,3 ng/uL


16.08.2012

Preparing the pRHA primers we received

  • 100uM stock in the tube and then and ependorff of 10uM

PCR of pRHA

  • We don't have a lot of pRHA left (synthetized oligo) so we will amplify pRHA from pRG116 and pRG113
  • phusion DNA pol was used

Verification gel of pRHA

  • 2% agarose gel, 100bp ladder
  • expected size of fragement: 165bp
  • 6 reactions + 2 ladder + 1 neg control
  • results: products in all tubes gave appropriate migration

PCR purification

  • pRG114 A: 74,4ng/uL
  • pRG114 B: 78,7ng/uL
  • pRG114 C: 82,3ng/uL
  • pRG114 D: 70,6ng/uL
  • pRG114 E: 71,6ng/uL
  • pRG114 F: 63,7ng/uL

Preparing the tubes containing the 2gblocks (RBS+FseI)

  • We add 20uL of H2O and get a concentration of 10ng/uL
  • We cannot do the digestion yet to then ligate the two fragments together because we do not have the AvaII enzyme yet

15.08.2012

Sending parts for sequencing

  • parts pRG106 to pRG113
  • Results: were all consistent


14.08.2012

Verification gel

  • 1% agar gel, 1kb ladder, 48 reactions + 1 neg control
  • expected sizes:
    1. pBAD+RBS+IsceI+PSB3C5 (pRG106): 1151
    2. pBAD+RBS+GFP+PSB3C5 (pRG107): 1294
    3. pBAD+RBS+RFP+PSB3C5 (pRG108): 1118
    4. pLac+RBS+IsceI+PSB3C5 (pRG109): 1076
    5. pLac+RBS+GFP+PSB3C5 (pRG110): 1219
    6. pLac+RBS+RFP+PSB3C5 (pRG111): 1043
    7. pRHA+RBS+IsceI+PSB3C5 (pRG112): 1143
    8. pBAD+RBS+RFP+PSB3C5 (pRG113): 1110

Glycerol stock

  • 750uL from each tube of liquid culture + 250uL of 60%glycerol

MiniPrep

  • pRG106: 362,9ng/ul
  • pRG107: 314,3ng/ul
  • pRG108: 253,4ng/ul
  • pRG109: 236,6ng/ul
  • pRG110: 228,7ng/ul
  • pRG111: 347,2ng/ul
  • pRG112: 227,2ng/ul
  • pRG113: 289,7ng/ul

13.08.2012

Ligation results

  • Good results in each plate (a lot more colonies in the ligation plate than in the negative control), except in the plate containing (pRHA+RBS+GFP+pSB1C5) where there are no colonies at all (we will have to do this ligation again).
  • We will take 3 colonies per plate and run a control PCR and start a liquid culture.

Colony PCR

  • 3 colonies per plate
  • 48 reactions + 2 negative control = 50 eactions

Liquid culture

  • 3 colonies per plate (the same used to run the culture PCR)
  • 48 tubes + 1 neg control = 49 tubes
  • Antibiotic: Cm

09.08.2012

08.08.2012

Lost of plasmids experiment

  1. Pick a colony from transformation plate (see 06.08.2012) and start a liquid culture:
    • 5 ml of LB + 5 ul of Amp + 5 ul of Cm
  2. Wait while OD = 1.25
  3. Wash cells and add 10 ml LB without antibiotic.
  4. Divide it into 2 tubes:
    • 5 ml of LB with cells + 5 ul of Amp
    • 5 ml of LB with cells + 5 ul of Amp + 5 ul of IPTG
    Final OD of each LC is equal 0.6
  5. Overnight grow.
  6. Mesure OD (OD = 2.95 for both tubes)
  7. Dilute it (x3000)
  8. Plate 50 ul and 100 ul from each tube on 2 kinds of plate:
    • Amp;
    • Amp + Cm;
  9. Wait overnight to get results

Run a gel (Colony PCR results)

The first gel
The first gel
The second gel
The second gel
The third gel
The third gel
The fourth gel
The fourth gel
  • Gel: 0.7% of agarose;
  • Ladder: 1 kb (fermentase);
  • Order on the gel:
    • First gel:
      • Ladder
      • pRG.061 - pRG.068 (pBad + RBS + I-SceI + pSB1A3)
        • Expected size: 1185 bp
      • Ladder
      • pRG.069 - pRG.074 (pLac + RBS + I-SceI + pSB1A2)
        • Expected size: 1034 bp
      • Ladder
    • Second gel:
      • Ladder
      • pRG.075, pRG.076 (pLac + RBS + I-SceI + pSB1A2)
        • Expected size: 1034 bp
      • pRG.085 - pRG.088 (pBad + RBS + GFP + pSB1A3)
        • Expected size: 1328 bp
      • Ladder
      • pRG.089 - pRG.092 (pLac + RBS + GFP + pSB1A2)
        • Expected size: 1058 bp
      • pRG.101 - pRG.104 (pLac + RBS + RFP + pSB1A2)
        • Expected size: 1001 bp
      • Ladder
    • Third gel:
      • Ladder
      • pRG.075, pRG.076 (pLac + RBS + I-SceI + pSB1A2)
        • Expected size: 1034 bp
      • pRG.085 - pRG.088 (pBad + RBS + GFP + pSB1A3)
        • Expected size: 1328 bp
      • Ladder
      • pRG.089 - pRG.092 (pLac + RBS + GFP + pSB1A2)
        • Expected size: 1058 bp
      • pRG.101 - pRG.104 (pLac + RBS + RFP + pSB1A2)
        • Expected size: 1001 bp
      • Ladder


The size is right for all parts.

07.08.2012

Colony PCR

  • It was done 50 reactions:
    • Control reaction (without adding a template DNA).
    • We picked colonies from different plates:
Ligated constructs Constructs Transformation plates
2 ul 8 ul
pRG.003 + pRG.055
pBad + RBS + I-SceI + pSB1A3
pRG.061
pRG.062
pRG.063; pRG.064; pRG.065;
pRG.066; pRG.067; pRG.068;
pRG.037 + pRG.055
pLac + RBS + I-SceI + pSB1A2
pRG.069
pRG.070
pRG.071; pRG.072; pRG.073;
pRG.074; pRG.075; pRG.076;
pSB3C5 + pRG.055
RBS + I-SceI + pSB3C5
pRG.077
pRG.078
pRG.079; pRG.080; pRG.081;
pRG.082; pRG.083; pRG.084;
pRG.003 + pRG.007
pBad + RBS + GFP + pSB1A3
pRG.085
pRG.086; pRG.087; pRG.088;
pRG.037 + pRG.007
pLac + RBS + GFP + pSB1A2
pRG.089
pRG.090; pRG.091; pRG.092;
pSB3C5 + pRG.007
RBS + RFP + pSB3C5
pRG.093
pRG.094; pRG.095; pRG.096;
pRG.003 + pRG.037
pBad + RBS + RFP + pSB1A3
pRG.097
pRG.098; pRG.099; pRG.100;
pRG.037 + pRG.037
pLac + RBS + RFP + pSB1A2
pRG.101
pRG.102; pRG.103; pRG.104;
pSB3C5 + pRG.037
RBS + GFP + pSB3C5
pRG.105
pRG.106; pRG.107; pRG.108;

06.08.2012

Run a gel (Ligation results)

Order on the gel: ladder, pRG.053 - pRG.056, ladder, pRG.057 - pRG.060, control, ladder
Order on the gel: ladder, pRG.053 - pRG.056, ladder, pRG.057 - pRG.060, control, ladder
  • Expected size:
    • RBS + I-SceI (pRG.053 - pRG.056): 971 bp;
    • RBS + RFP (pRG.057 - pRG.060): 938 bp
  • Gel: 1% of agarose;
  • Ladder: 1 kb (fermentase);

Order on the gel: ladder, pRG.053 - pRG.056, ladder, pRG.057 - pRG.060, control, ladder


The size is right for all parts (pRG.056 - pRG.060).

Glycerol stock & Miniprep

Based on the gel result the next parts were choosen for the glyserol stock and Miniprep:

  • RBS + I-SceI: pRG.055 207.6 ng/ml;
  • RBS + RFP: pRG.057 193.0 ng/ml;

Digestion

Reagent Volume
RBS + I-SceI RBS + GFP RBS + RFP pBad pLac pSB3C5
Water (nuclease free)
27.5 ul
27 ul
27 ul
28 ul
24 ul
28.5 ul
10x Fast digest buffer
4 ul
4 ul
4 ul
4 ul
4 ul
4 ul
DNA
4.5 ul
5 ul
5 ul
4 ul
8 ul
3.5 ul
XbaI (Fast digest)
2 uL
2 uL
2 uL
-
-
2 uL
PstI (Fast digest)
2 uL
2 uL
2 uL
2 uL
2 uL
2 uL
SpeI (Fast digest)
-
-
-
2 uL
2 uL
-
Total volume:
40 ul
40 ul
40 ul
40 ul
40 ul
40 ul
  • Digestion time: 75 min.
  • Digestion temperature: 37°C.
  • Shaking: 400 rpm


Run a gel (Digestion)

Digestion results of RFP and RBS & pSB1A2
Digestion results of RFP and RBS & pSB1A2
  • Gel: 1% of agarose;
  • Ladder: 1 kb (fermentase);
  • Order on the gel:
    • First gel:
      • Digestion of pRG.055 (RBS + I-SceI + pSB1A2)
        • Expected size: 759 bp
      • Ladder
      • Empty
      • Digestion of pRG.007 (RBS + GFP + pSB1A2)
        • Expected size: 902 bp
      • Ladder
      • Digestion of pRG.057 (RBS + RFP + pSB1A2)
    • Second gel:
      • Digestion of pSB3C5
        • Expected size: 2720 bp
      • Ladder
      • Empty
      • Digestion of pRG.003 (pBad + pSB1A3)
        • Expected size: 2275 bp
      • Ladder
      • Digestion of pRG.037 (pLac + pSB1A2)
        • Expected size: 2124 bp

All parts are good to be cutted and purified.

Purification results

Purification of the PCR product digestion and the gel purification was done by following stendard protocol:

  • pRG.055 (RBS + I-SceI + pSB1A2) digested with X and P: 2.6 ng/ml
  • pRG.007 (RBS + GFP + pSB1A2) digested with X and P: 4.0 ng/ml
  • pRG.057 (RBS + RFP + pSB1A2) digested with X and P: 2.2 ng/ml
  • pRG.003 (pBad + pSB1A3) digested with S and P: 4.5 ng/ml
  • pRG.037 (pLac + pSB1A2) digested with S and P: 11.4 ng/ml
  • pSB3C5 digested with X and P: 5.2 ng/ml

Ligation

We are ligating the next constructs:

  • Insert:
    • pRG.055 (RBS + I-SceI + pSB1A2) digested with X and P;
    • pRG.007 (RBS + GFP + pSB1A2) digested with X and P;
    • pRG.057 (RBS + RFP + pSB1A2) digested with X and P;
  • Vector:
    • pRG.003 (pBad + pSB1A3) digested with S and P;
    • pRG.037 (pLac + pSB1A2) digested with S and P;
    • pSB3C5 digested with X and P;


Vector
0.5 ul
Insert
8 ul
Water (nuclease free)
0 ul
T4 Ligase buffer (Fermentas)
1.0 uL
T4 Ligase Enzyme (Fermentas)
2 uL
Total volume:
10 ul

Transformation

1) Transformation of the ligation product was done by following the standard protocols:


2) It was transformed two plasmids of TUDelft for Lost of plasmids experiment:

  • K175041:
    • Construct: pLac + RBS + I-SceI
    • Resistance: AmpR
  • K175027:
    • Construct: RS
    • Resistance: CmR

Transformation was done by following standard protocol:

2 ul of K175041 and 2 ul of K175041 was transformed.

05.08.2012

Transformation results

Transformation of the ligation product is successful. Three liquid cultures are started:

  • LB: 6 ml * 8 = 48 ml ≈ 50 ml;
  • All colonies are AmpR (50 ul);

Colony PCR

  • It was done 10 reactions:
    • We picked 2 colonies from each plate.
    • Control reaction (without adding a template DNA).
Constructs Transformation plate
2 ul 8 ul
I-SceI + RBS/psB1A2
pRG.053
pRG.054
pRG.055
pRG.056
RFP + RBS/psB1A2
pRG.057
pRG.058
pRG.059
pRG.060

04.08.2012

Digestion of PCR product

  • Digestion of I-SceI which was amplified by PCR from SMR 6316 stain (pRG.024).
  • Pipetting instruction:
Purified PCR product
30 ul
H20
4 ul
10x Fast digest buffer
4 ul
XbaI (Fast digest)
2 uL
PstI (Fast digest)
2 uL
Total volume:
42 ul
  • Digestion time: 60 min.
  • Digestion temperature: 37°C.

Digestion of Vectors

Reagent Volume
RFP RBS + pSB1A2
Water (nuclease free)
18 ul
26.5 ul
10x Fast digest buffer
4 ul
4 ul
DNA
14 ul
5.5 ul
XbaI (Fast digest)
2 uL
-
PstI (Fast digest)
2 uL
2 uL
SpeI (Fast digest)
-
2 uL
Total volume:
40 ul
40 ul
  • Digestion time: 75 min.
  • Digestion temperature: 37°C.


Run a gel (Digestion)

Digestion results of RFP and RBS & pSB1A2
Digestion results of RFP and RBS & pSB1A2
Digestion results of RFP and RBS & pSB1A2
Digestion results of RFP and RBS & pSB1A2
  • Gel: 1% of agarose;
  • Ladder: 1 kb (fermentase);
  • Order on the gel:
    • Digestion of RFP(pRG.050)
    • Digestion of RFP(pRG.052)
      • Expected size: 705 bp
    • Ladder
    • Digestion of RBS & pSB1A2 (pRG.021)
    • Digestion of RBS & pSB1A2 (pRG.022)
      • Expected size: 2082 bp

For RFP we decided to cut only pRG.052, for RBS & pSB1A2 both.

Purification results

Purification of the PCR product digestion and the gel purification was done by following a stendard protocol:

  • I-SceI (X & P): - ng/ml
  • RFP (X & P): - ng/ml
  • RBS & pSB1A2: 16.6 ng/ml


Ligation

We are ligating the next constructs:

  • Insert:
    • I-SceI
    • RFP
  • Vector:
    • RBS & pSB1A2
Vector
0.5 ul
Insert
8 ul
Water (nuclease free)
0 ul
T4 Ligase buffer (Fermentas)
1.0 uL
T4 Ligase Enzyme (Fermentas)
2 uL
Total volume:
10 ul


Transformation of the ligation product

Transformation of the ligation product was done by following the standard protocols:

03.08.2012

Miniprep & Glycerol

Miniprep and glycerol stock of liquid culture from yesterday (RFP, E1010):

  • pRG.050: 35.1 ng/uL
  • pRG.051: -
  • pRG.052: 68.8 ng/uL

Colony PCR (RFP, E1010)

  • It was done 5 reactions:
    • We picked bacteria from each plate (pRG.050 - pRG.052).
    • Control reaction (without adding a template DNA).
  • Expected size: 995 bp
  • Extension time: 1 min 20 s

Run a gel (Colony PCR of RFP)

Order on the gel: ladder, pRG.050, pRG.051, pRG.052, control, empty, ladder
Order on the gel: ladder, pRG.050, pRG.051, pRG.052, control, empty, ladder

Expected size: 82 bp;

  • Gel: 1% of agarose;
  • Ladder: 1 kb (fermentase);
  • Order on the gel: ladder, pRG.050, pRG.051, pRG.052, control, empty, ladder;

All three parts are good.

02.08.2012

RFP transformation results

Transformation is successful. Three liquid cultures are started.

01.08.2012

Transformation of RFP

We decided transform an RFP for our control constructs:

  • E1100 - highly engineered mutant of RFP from Discosoma striata
    • Distribution: 2012 kit, plate 1
    • Well: 18 F

30.07.2012

Tekan experiment

29.07.2012

Amplification of I-SceI

It was used Phusion high-fidelity DNA polimerase.

  • Size of I-SceI: 770 bp
  • Pipetting instruction:
Water (Nuclease free)
33.5 ul
5x Phusion HF Buffer
10 ul
10 mM dNTPs
1 uL
Forward primer
2.5 uL
Reverse primer
2.5 uL
Template DNA
Pick a colony
Fusion DNA polimerase
0.5 ul
Total volume:
50 ul
  • It was done 7 reactions:
    • We picked bacteria from each of 6 glycerol stocks of SMR 6316 strain (pRG.023 - pRG.028).
    • Control reaction (without adding a template DNA).
  • Cycling instructions:
Step Temperature, °C Time Number of cycles
Initial denaturation
98
10 min
1
Denaturation
98
10 s
20
Annealing
50
30 s
Extension
72
30 s
Final Extension
72
10 min
1
End
8
Forever
1

pLac digestion

  • Digestion plasmids from miniprep stock:
    • pRG.035 (114 ng/uL)
    • pRG.037 (152 ug/uL)
    • pRG.039 (143 ng/uL)
  • Pipetting instructions:
Water (Nuclease free)
20 ul
10x Fast Digest Buffer
4 ul
DNA
12 ul
EcoRI
2 ul
SpeI
2 ul
Total volume:
40 ul
  • 10 min: 37°C

Run a gel (Digestion results)

  • Expected size: 82 bp;
  • Gel: 2% of agarose;
  • Ladder: 100 bp (fermentase);
  • Order on the gel: ladder, pRG.035, pRG.037, pRG.039, ladder;

Gel wasn't homogeneous, but we are sure, that is size of sequence is right. So, we decided to cut and purify it.

Tekan Experiment (Start a liquid culture)

Goal: Approve that parts from distribution plates which we are planning to use as our control plasmids are working.

  • List of parts:
  1. pRG.009, pRG.010: pLacI + RBS (B0030) + GFP + TT + pSB1A2;
  2. pRG.013, pRG.014: pLacI + RBS (B0034) + RFP + T + pSB1A3;
  3. pRG.011, pRG.012: pBad + RBS (B0034) + GFP + pSB1AK3;
  4. pRG.015, pRG.016: pBad/araC + RBS (B0034) + GFP + TT + pSB1A3;
  5. pRG.046, pRG.047: pBad + RBS (B0032) + GFP + pSB1A3;


  • pLac is induced by IPTG:
    • 5 ml of LB + 5 ul of IPTG + 5 ul of Amp
  • pBad is induced by Arabinose:
    • 4.5 ml of LB + 0.5 mL of 20% Arabinose solution + 5 ul of Amp

Run a gel (I-SceI amplification)

  • Expected size: 770 bp;
  • Gel: 1% of agarose;
  • Ladder: 1 kb (fermentase);
  • Order on the gel: ladder, pRG.023 - pRG.026, ladder, pRG.027, pRG.028, negative control, ladder;

We have good results only for pRG.024.

27.07.2012

PCR reults from 26.07.2012

PCR reults to check size of ligation products from 26.07.2012
PCR reults to check size of ligation products from 26.07.2012


Run a gel (from left to right):

  • Ladder (1kb, Fermentas).
    Positions: 1, 10, 17;
  • Results of ligation: I13453 (pBad, Miniprep Stock Name: pRG.003, pRG.004) and B0032 (RBS, Miniprep Stock Name: pRG.022)
    Positions: 2, 3, 4, 5, 6, 7;
    Expected size: 389 bp
  • Results of ligation: I13453 (pBad, Miniprep Stock Name: pRG.003) and E0240 (RBS + GFPmut3 + TT, Miniprep Stock Name: pRG.007, pRG.008)
    Positions: 8, 9, 11, 12, 13, 14;
    Expected size: 1 252 bp
  • Positive control: R0011(pLac)
    Positions: 15;
    Expected size: 293 bp
  • Negative control: Empty tube
    Positions: 16;
    Expected size: 0 bp

Comments: It seems all results except position 14 are acceptable. We choose positions 4 and 6 for pBad + RBS, 11 and 12 for pBad + RBS + GFPmut3 + TT to make glycerol stock and miniprep.

Purification of the gel parts we cut from the gel yesterday: RBS/GFP (X,P), pSB3C5 (E,S), pLAC (NOTI)

We used a standard protocol. At the end we obtained the following concetnrations:

RBS/GFP (X,P): 49.9ng/uL
pSB3C5 (E,S): 27.3ng/uL
pLAC (NotI): 263.8ng/uL

Testing the fluorescence of the liquid culture I did yesterday

Making glycerol stock of ligation p0022+p003 and p007+p003

  • see exel file for naming of tubes
  • We used the PCR/gel results to see which tube to glycerol and miniprep. we too product that was in tube 3,5,9, 10
  • Standard protocol

Minipreping p0022+p003 and p007+p003

  • Standart protocol.
  • In the end we obtained:
pRG0044: (p022+p003): 156.9ng/uL
pRG0045 (p007+p003): 59.0ng/uL
pRG0046 (p007+p003): 66.3ng/uL

NB: only one tube instead of 2 because I made a mistake and used the same pipete twice which contaimnated my second tube for (p022+p003)

26.07.2012

Digestion of pLAC (NOTI), pSB3C5 (E, S), E0240 (X,P)

  • We digested pLac with NOT I because previous gels were not conclusiive when we digested it with standard biobrick enzymes (it appeared too big). However, later, we figured out that this was due to an error with the ladder.However, at that date, we did not know that and decided to digest the part with NOT I (there are two NOT I restriction sites in between E,X and S,P) in case something was wronf with the standard biobrick restriction sites.
  • We digested pSB3C5 with E and S (so we could put pbad that was already digested with E and S in the standard vector, and gain time when the gBLOCKs arrive as one cloning step would already have been done).
  • We digested E0240 (RBS/GFP) with X and P so we could put in in the pLac/pSB1C3 vector if that first stepped worked. This is also another way to achieve the 2 step ligation we described yesterday. We find it safer to try various ways in parallel as ligation often does not work the first time. However, we did not do that with RBS (digest it with X and P) because it is too small and would have been hard to purify after digestion.

The digestion was done using 10uL of DNA each time and a standard protocol (however i messed up with the units and ended up putting way too much enzymes: 5uL in total per tube instead of 4uL, in a total volume of 50uL). Then I left the tube way too long (3hours) before running the gel. However, the gel showed satisfying results (we had the expected sizes for each part).

Conclusion: these digestions were successful.

Running a gel for these digestions

Expected sizes:

E0240: 902bp
pSB3C5: 2721
pLac: 83
  • We runned E0240 and pSB1C3 on a 1.2% agarose gel
  • We runned pLac on a 2% agarose gel

(PUT PICTURE)

  • The size were the ones we expected. We cut the pieces and put them in a 2uL ependorf. TO be purified tomorow.

Preparing more pSB3C5 out of glycerol stock

  • We started liquid culture (C resistant) on this day
  • The next day nothing had grown in the tube so we could not miniprep.
  • We will have to start more liquid culture another day.

Preparing liquid cultures of the ligation made on the 24.07

We made 20 tubes:

we had 3 plates for each ligation (so a total of 6 plates)
we took two colonies from each plate
for the ligation product containing PLac/GFP we made two tubes for each colonies. One tube containing arabinose 0.2% and one tube containing 2% glucose. Arabinose is supposed to induce the promoter (and so our cells should be fluorescent) and glucose to inhibit it.

RESULT: The cells that had grown over night in the tubes containing ara were fluorescent and the one in the tubes containg glucose were not fluorecent. However, the control (pLac/RBS) was also fluorescent. Maybe the plasmid is fluorescent? But then maybe there was a pb with the cells in glucose? To investigate furtehr!

PCR of the colonies we made liquid culture

  • we do this in order to know if the ligation really worked and which tube to take cells from to do the miniprep and glycerol.
  • We runned the PCR overnight. Gel will be runned the next day on 27.07

25.07.2012

We want to clone the following things:

pBAD + RBS + pSB3C5
pBAD + RBS/GFP + pSB3C5

This requires 2 steps:

  1. We ligate pBAD with RBS and pBAD with RBS/GFP
  2. We ligate the product of our first ligation (pBAD/RBS and pBAD/RBS/GFP) with pSB3CS

NB: we want to put our parts in the pSB3C5 vector because it is the standard final vector we decided to use for the whole construct.

Ligation of pBAD with RBS and ligation of pBAD with RBS/GFP

  • We ligated p003 (pBAD) with p022 (RBS). To respect the 3:1 insert vector ratio, we took 1uL of p022 and 4.9 uL of p003. We then followed a stadard protocol.
  • We ligated p003 (pBAD) with p0007 (RBS). To respect the 3:1 insert vector ratio, we took 1uL of p007 and 5.3 uL of p003. We then followed a stadard protocol.

After the ligation, we put the tubes at 65°c for 10 minutes in order to inactivate the ligation enzymes before transformation.

Transforming NEB turbo cells with our two ligations

  • In our tubes, we had 10uL of ligation product.
  • We took 3 uL and put it in a tube containing 20uL of competent NEB tubo cells. We did this step in total 3 times per ligation product.
  • We then heat shocked our cells and plated them following a standard protocol.
  • Our positive control was R0011 (pLac from pRG.018)


Colony PCR

Goal: Check their size

  1. Gently vortex and briefly centrifuge PCR Master Mix (2X) after thawing.
  2. Place a thin-walled PCR tube on ice and add the following components for each 50ul reaction:
  3. Chemical Volume
    PCR Master Mix (2X)
    25 ul
    Forward primer
    2.5ul
    Reverse primer
    2.5ul
    Template DNA
    Put a loop
    Water, nuclease-free
    to 50ul
    Total volume:
    50ul
  4. Gently vortex the samples and spin down.
  5. When using a thermal cycler that doesn't contain a heated lid, overlay the reaction mixture with 25ul of mineral oil.
  6. Perform PCR using the recommended thermal cycling conditions outlined below:
  7. Step Temperature, °C Time Number of cycles
    Initial denaturation
    95
    10 min
    1
    Denaturation
    95
    30 s
    30
    Annealing
    50
    30 s
    Extension
    72
    1 min
    Final Extension
    72
    10 min
    1
    End
    8
    Forever
    1


  • Comments: LID = 100°C


Gel Results

First Gel (Wrong picture)
First Gel (Wrong picture)
Second Gel
Second Gel

First gel (from left to right):

  • Ladder (1kb).
    Positions: 1, 8, 15;
  • pRG.023 - pRG.028 (pBad & I-SceI, primers was used: iRG.001, iRG.002)
    Positions: 2 - 7;
    Expected size: 1897 bp
  • pRG.023, pRG.024 (pBad & I-SceI, primers was used: iRG.001, iRG.002)
    Positions: 9, 10;
    Expected size: 1897 bp
  • pRG.025 - pRG.028 (I-SceI, primers was used: iRG.003, iRG.002)
    Positions: 11 - 14;
    Expected size: 293 bp

Second gel (from left to right):

  • Ladder (1kb).
    Positions: 1, 8, 15;
  • pRG.029 - pRG.031 (K175027, primers was used: VF, VR2)
    Positions: 2 - 4;
    Expected size: 346 bp
  • pRG.032 - pRG.034 (K175041, primers was used: VF, VR2)
    Positions: 5 - 7;
    Expected size: 1430 bp
  • pRG.035 - pRG.036 (pLac from the first transformation plate, primers was used: VF, VR2)
    Positions: 9 - 10;
    Expected size: 174 bp
  • pRG.037 - pRG.038 (pLac from the sequencing tube, primers was used: VF, VR2)
    Positions: 11 - 12;
    Expected size: 174 bp
  • pRG.039 - pRG.040 (pLac from the SG stock, primers was used: VF, VR2)
    Positions: 13 - 14;
    Expected size: 174 bp

Comments: It seems all results acceptable. Note: it is not fermentase leddar!


24.07.2012

Preparing PCR tubes containg the TU DELF parts for sequencing

We sent the two parts we received from TU DELFT K175027 (IsceI restriction site) and K175041 (pLac controlled IsceI endonuclease) to sequencing in order to make sure these parts were the correct one. We sent the PCR tubes (made the previous day) and gatc purified them themselves before sequencing.

We received the results two days later. The sequencing results were consistant.

MiniPreping some more pRG005 & pRG006

  • There was no more pRG005 (pLac) & pRG006 left from the miniprep we made on 08.07.2012. Therefore we hade to make some more.
  • We followe a stadard protocol. At the end we ended upwith the following concentrations inthe tubes:
pRG.005 = 190.1 ng/uL (260/280 ratio = 1.90)
pRG.006 = 254.9 ng/uL (260/280 ratio = 1.90)

Purification of pBAD p003 (E,S), pSB1C3 (E,P), RBS B0032 p022 (E,X), RBS/GFP p003 (E,X) from gel

  • The previous day, we had digested these parts, then migrated them on a gel and cut them out of the gel. They were then placed in 2uL ependorf tubes.
  • Now we want to purify these part so we can use it for a 2 step ligation: ligation of pBAD with RBS/GFP and RBS (step 1) and then put everything in pSB3C5 (step 2).
  • We followed a standard protocol. At the end we obtained purified pieces at the following concentrations:
pBAD (p003) purified after digestion (E,S) = 12.7ng/uL
pSB1C3 purified after digestion (E,P) = 43.5ng/uL
RBS B0032 (p022) after digestion with (E,X) = 20.8ng/uL
RBS/GFP (p003) after digestion with (E,X) = 22.4ng/uL

23.07.2012

Digestion of pLac with E and P, like Julianne and Aishah did; Also digestion of pSB3C5 with E and P to put the pLac inside


Digestion of pLac again gave strange results: band at appx. 250-300bp instead of around 70.
Digestion of plasmid pSB3C5 successful: cut out the fragment of around 3kb (expected size around 2.7kb so digestion successful)

Gel_pRG_2307_digestion2.png
Gel_pRG_2307_digestion2.png

PCR of K175027 and K175041

We received these parts from anotehr igem team

K175027: it is the restriction site for IsceI. Size: 30bp K175041: pLacI controlled IsceI homing endonuclease generator (IsceI ha an LVA degradation tag). Expected size: 1114bp

We made a PCR using 1uL of DNA from these 2 tubes. The protocol was exactly the same then the one described just below.

  • Gel results:


The ISceI site (K175027) should be 342 bp after PCR. Result consistent.
The pLac/ISceI (K175041) should be 1426bp after PCR. Result consistent.

IGEM_Paris_Bettencourt_2012_230712_Delft_parts.png
IGEM_Paris_Bettencourt_2012_230712_Delft_parts.png

Gel of miniPrep digestion I13453 and R0011

Digestion of miniPrep I13453 and R0011

Gel of PCR BBa_I13453 (pBad) and BBa_R0011 (pLac)

PCR of miniprep BBa_I13453 (pBad) and BBa_R0011 (pLac)

Since all our gels for these parts were inconsistant, put the sequencing was consistent, we decided to make yet anotehr gel, hence the PCR.

Biobrick name Taken from tube Name of PCR tube Size of fragment
BBa_I13453 (pBAD) pRG 003 1
BBa_I13453 (pBAD) pRG 004 2
BBa_R0011 (pLac) pRG 005 3
BBa_R0011 (pLac) pRG 006 4


  1. Gently mix PCR Master Mix (2X) after thawing.
  2. Place a thin-walled PCR tube on ice and add the following components for each 50ul reaction:
  3. Chemical Volume
    PCR Master Mix (2X)
    25 uL
    Forward primer (VF2)
    2.5uL
    Reverse primer (VR)
    2.5uL
    Template DNA
    1 uL
    Water, nuclease-free
    19 uL
    Total volume:
    50ul
  4. Gently mix the samples.
  5. Perform PCR using the recommended thermal cycling conditions outlined below:
  6. Step Temperature, °C Time Number of cycles
    Initial denaturation
    95
    2 min
    1
    Denaturation
    95
    30 s
    25
    Annealing
    50
    30 s
    Extension
    72
    1 min
    Final Extension
    72
    10 min
    1
    End
    8
    Forever
    1


  • Comments: LID = 100°C

22.07.2012

Glycerol Stock

Part Description Plasmid Backbone Size, bp Colony Stock name
BBa_K098991
A constitutive GFP reporter driven by the cI promoter + NEB Turbo
pSB1A2
933
T1
pRG.019
T2
pRG.020
BBa_K093012
Strong constitutive promoter - RFP + NEB Turbo
pSB1A2
767
T3
pRG.021
T4
pRG.022
BBa_B0032
RBS.3 (medium) -- derivative of BBa_0030
pSB1A2
13
T5
pRG.023
T6
pRG.024

21.07.2012

Transformation results

Successful transformation:

Unsuccessful transformation:

Start Culture for miniprep and glycerol

  • We pick 2 colonies from each plate with successful transformation.
  • Add it to 6 ml of LB + Ampicilline (6 tubes: T1, T2, T3, T4, T5, T6).
  • Overnight incubation.

Colony PCR of BBa_K098991, BBa_K093012 and BBa_B0032

  1. Gently vortex and briefly centrifuge PCR Master Mix (2X) after thawing.
  2. Place a thin-walled PCR tube on ice and add the following components for each 50ul reaction:
  3. Chemical Volume
    PCR Master Mix (2X)
    25 ul
    Forward primer (VF2)
    2.5ul
    Reverse primer (VR)
    2.5ul
    Template DNA
    Put a loop
    Water, nuclease-free
    to 50ul
    Total volume:
    50ul


  4. Gently vortex the samples and spin down.
  5. When using a thermal cycler that doesn't contain a heated lid, overlay the reaction mixture with 25ul of mineral oil.
  6. Perform PCR using the recommended thermal cycling conditions outlined below:
  7. Step Temperature, °C Time Number of cycles
    Initial denaturation
    95
    10 min
    1
    Denaturation
    95
    30 s
    25
    Annealing
    50
    30 s
    Extension
    72
    1 min
    Final Extension
    72
    10 min
    1
    End
    8
    Forever
    1


  • Comments: LID = 100°C

20.07.2012

Biobrick Retrieval Protocol was used. List of retrieval parts:

Part Description Location in a kit Plasmid Backbone
BBa_B0032
RBS.3 (medium) -- derivative of BBa_0030
2012 Kit Plate 1, well 2I
pSB1A2
BBa_K098991
A constitutive GFP reporter driven by the cI promoter.
2012 Kit Plate 3, well 1C
pSB1A2
BBa_R0051
promoter (lambda cI regulated).
2012 Kit Plate 1, well 6K
pSB1A2
BBa_K093012
Strong constitutive promoter - RFP.
2012 Kit Plate 3, well 13M
pSB2K3

Heat Shock Transformation

  • Parts from the list above were transformed using Heat Shock Transformation protocol:
    20ml of competent NEB Turbo cells + 2ml of the plasmids.
  • The remaining plasmids (8ml) were stored at -20°C.

16.07.2012

Designing primers for IsceI and pBAD+IsceI from SMR6316 cells

14.07.2012

Restriction Digestion

  • The following parts were digested with EcoRI and PstI restriction enzimes:
Part Description Plasmid Backbone Size, bp Stock name
BBa_E0240
RBS (B0032) + GFPmut3 + TT
pSB1A2
876
pRG.007 & pRG.008
BBa_K117004
pLacI + RBS (B0030) + GFPmut3 + TT
1086
pRG.009 & pRG.010
BBa_I746902
pBad + RBS (B0034) + GFPmut3 (His-tagged)
2011
pRG.011 & pRG.012
BBa_J04450
pLacI + RBS (B0034) + mRFP + T
1069
pRG.013 & pRG.014
BBa_I13540
pBad/arac + RBS (B0034) + GFP (E0040) + TT
2093
pRG.015 & pRG.016
BBa_R0011
pLacI
55
pRG.017 & pRG.018


  • The following protocol was used:
Chemical Volume
Water
14 ul
Green buffer
2ul
DNA
2ul
EcoRI
1ul
PstI
1ul


  • After 10 min at 37°C (???)

Gel

From left to right (expected size): ladder, pRG.007 - pRG.018, ladder
From left to right (expected size): ladder, pRG.007 - pRG.018, ladder


We run a gel to control colony PCR results. To do the PCR we use the next primers:

  • Forward primer: VF2
  • Reverse primer: VR


From left to right:

  1. Ladder = 5 ml;
  2. pRG.007 (GFP, BBa_E0240) = 5ml + 1ml of dye = 6ml. Expected size: 720bp
  3. pRG.008 (GFP, BBa_E0240) = 5ml + 1ml of dye = 6ml. Expected size: 720bp
  4. pRG.009 (pBad, BBa_K117004) = 5ml + 1ml of dye = 6ml. Expected size: 130bp
  5. pRG.010 (pBad, BBa_K117004) = 5ml + 1ml of dye = 6ml. Expected size: 130bp
  6. pRG.011 (pBad, BBa_I746902) = 5ml + 1ml of dye = 6ml. Expected size: 130bp
  7. pRG.012 (pBad, BBa_I746902) = 5ml + 1ml of dye = 6ml. Expected size: 130bp
  8. pRG.013 (pBad, BBa_J04450) = 5ml + 1ml of dye = 6ml. Expected size: 130bp
  9. pRG.014 (pBad, BBa_J04450) = 5ml + 1ml of dye = 6ml. Expected size: 130bp
  10. pRG.015 (pBad, BBa_I13540) = 5ml + 1ml of dye = 6ml. Expected size: 130bp
  11. pRG.016 (pBad, BBa_I13540) = 5ml + 1ml of dye = 6ml. Expected size: 130bp
  12. pRG.017 (pBad, BBa_R0011) = 5ml + 1ml of dye = 6ml. Expected size: 130bp
  13. pRG.017 (pBad, BBa_R0011) = 5ml + 1ml of dye = 6ml. Expected size: 130bp
  14. Ladder = 5 ml;


13.07.2012

Purification (MiniPrep)

Part Description Plasmid Backbone Size, bp Colony Stock name Concentration, ng/ul
BBa_E0040
GFP
pSB1A2
720
T1
pRG.001
277.4
T2
pRG.002
233.3
BBa_I13453
pBad
pSB1A3
130
T3
pRG.003
246.4
T4
pRG.004
191.6
BBa_R0011
pLac
pSB1A2
55
T5
pRG.005
279.8
T6
pRG.006
215.4


Glycerol stock

  • We make two tubes per liquid culture tube. One that will be stored at -20°C and another one that will be stored at -80°C
  • In each tube we added:
  1. 750uL of solution from the culture tube
  2. 250 uL of glycerol 60%
Biobrick name Colony Tube name
BBa_E0240 1 pRG007
BBa_E0240 2 pRG008
BBa_K117004 1 pRG009
BBa_K117004 2 pRG010
BBa_I746902 1 pRG011
BBa_I746902 2 pRG012
BBa_J004450 1 pRG013
BBa_J004450 2 pRG014
BBa_I13540 1 pRG015
BBa_I13540 2 pRG016

12.07.2012

Results of sequencing

Sequencing results for PLac and PBAD were consistent.

Start Culture of E0240, K117004, I746902, J04450, I13540, I13540, Control+ R0011 trasformant

  • Using two colonies that formed (colonies 1 and 2) in each plate.
  • 6 mL of LB + Ampicilline (for E0240, K117004, I746902, I13540, I13540, Control+ R0011)
  • 6 mL of LB + Kan (for J04450)
  • Incubate over night

Gel

We made a 1% agarose gel

  1. Mix 50mL of BET (0.5%) with 0.5g of agarose
  2. Put in the microwave 1.30min and then an additional 20s (till the solution is transparent)
  3. Wait a little bit till it cools down (no more steam)
  4. Pull in a big plate (to make a big size gel)
  5. Wait 30min
  6. For each PCR tube: Mix 1uL of die with 5uL of solution from the PCR tube
  7. Put this mix in gel wells
  8. Wait for migration (approx 30min). Voltage: 100V
  9. Put the gel in an ethidium bromide bath for 10min
  10. Rince in H2O destain for 5min
  11. Take picture

From left to right, we've put the following parts in well:

gel_14_07_2012.gif
gel_14_07_2012.gif
  1. Ladder (1kb)
  2. E0240 colony 1 (pcr tube 1)
  3. E02410 colony 2 (pcr tube 2)
  4. Ladder 1kB
  5. K117004 colony 1 (pcr tube 3)
  6. K117004 colony 2 (pcr tube 4)
  7. I746902 colony 1 (pcr tube 5)
  8. I746902 colony 2 (pcr tube 6)
  9. J04450 colony 1 (pcr tube 7)
  10. J04450 colony 2 (pcr tube 8)
  11. I13540 colony 1 (pcr tube 9)
  12. I13540 colony 2 (pcr tube 10)
  13. Control+ R0011 colony 1 (pcr tube 11)
  14. Control+ R0011 colony 2 (pcr tube 12)
  15. K117004 colony 1 (pcr tube 3)
  16. Ladder 1kB

Gel Analysis: We do not see any parts. Something probably went wrong with the PCR. To check that our parts are well expressed, and are the correct parts we will tcheck it in another way: digestion of our minipreped plasmids and then gel (see 14.07.2012)

PCR of E0240, K117004, I746902, J04450, I13540, I13540, Control + R0011

  • Protocol from 07.07.12
  • DNA taken by picking colony from plates (nothing had grown in plate containing J09250)
Biobrick name Colony Tube n° Size of fragment
BBa_E0240 1 1 1076
BBa_E0240 2 2 1076
BBa_K117004 1 3 12286
BBa_K117004 2 4 12286
BBa_I746902 1 5 2311
BBa_I746902 2 6 2311
BBa_J004450 1 7 1269
BBa_J004450 2 8 11269
BBa_I13540 1 9 1269
BBa_I13540 2 10 1269
Control + (R0011) 1 11 255
Control + (R0011) 1 12 255

11.07.2012

Sequencing pLac and pBAD

  • We are going to sequence pBAD (tubes pRG003 and pRG004) and pLac (tube pRG005).
    We want to sequence pBAD because the band we obtained with the gel was not the expected size.
    We want to sequence pLac becaue we obtained it from Antoine D, and many people will use it, so we want to make sure its the good part.
  1. For sequencing,we need have 30uL (next time quantity should be 20uL) at a concentration of 50ng/uL (any concentration between 30 and 100ng/uL is good) of our plasmids in a 1.5mL eppendorf
  2. The primers we used are VR and VF2 at a concentration of 10uM
Sequencing results were consistant
Biobrick name Name of tube in which it can be find & Concentration (ng/uL) Name of tube we are making & Concentration (ng/uL) Qt that needs do be taken from existing tube(uL) Qt of DNAse free water to add (uL)
BBa_I13453 pRG003 & C= 246.4 pRG003.s & C= 30 6.1 23.9
BBa_I13453 pRG004 & C= 191.6 pRG004.s & C= 30 7.8 22.2
BBa_R0011 pRG005 & C= 279.8 pRG005.s & C= 30 5.4 24.6
  • We sent our tubes to be sequenced by gatc [1]

Biobrick Retrieval

Biobrick Retrieval Protocol was used. List of retrieval parts:

Part Description Location in a kit Plasmid Backbone
BBa_E0240
GFP generator: RBS (B0032)+ GFPmut3 + TT)
2012 Kit Plate 1, well 12M
pSB1A2
BBa_J09250
pLacIq + RBS (B0032)+ GFPmut3 + TT
2012 Kit Plate 2, well 9B
pSB2K3
BBa_K117004
pLacI + RBS (B0030) + GFPmut3 + TT
2012 Kit Plate 2, get 14J
pSB1A2
BBa_I13540
pBAD + RBS (B0034) + GFPmut3 + TT
iGEM 2007 Parts Kit Plate 2, well 17L
pSB1A3
BBa_I746902
pBAD + RBS (B0034) + GFPmut3 (His_tagged)
2012 Kit Plate 3, well 16F
pSB1AK3
BBa_J04450
RFP Coding Device: pLacI + RBS (B0034) + mRFP + TT
2012 Kit Plate 1, well 1G
pSB1A3
BBa_R0011
pLac (positive control)
Antoine stock
pSB1A2

Heat Shock Transformation

  • Parts from the list above were transformed using Heat Shock Transformation protocol:
    20ml of competent NEB Turbo cells + 2ml of the plasmids.
  • The remaining plasmids (8ml) were stored at -20°C.

08.07.2012

Glycerol Stock

Part Description Plasmid Backbone Size, bp Colony Stock name
BBa_E0040
GFP + NEB Turbo
pSB1A2
720
T1
pRG.001
T2
pRG.002
BBa_I13453
pBad + NEB Turbo
pSB1A3
130
T3
pRG.003
T4
pRG.004
BBa_R0011
pLac + NEB Turbo
pSB1A2
55
T5
pRG.005
T6
pRG.006

Gel

From left to right (expected size): ladder, pRG.001 (720bp), pRG.002 (720bp), pRG.003 (130bp), pRG.003 (130bp), ladder
From left to right (expected size): ladder, pRG.001 (720bp), pRG.002 (720bp), pRG.003 (130bp), pRG.003 (130bp), ladder

We run a gel to control colony PCR results. To do the PCR we use the next primers:

  • Forward primer: VF2
  • Reverse primer: VR


From left to right:

  1. Ladder = 5 ml;
  2. T1 (GFP, BBa_E0040) = 6ml + 1ml of dye = 7ml. Expected size: 720bp
  3. T2 (GFP, BBa_E0040) = 6ml + 1ml of dye = 7ml. Expected size: 720bp
  4. T3 (pBad, BBa_I13453) = 6ml + 1ml of dye = 7ml. Expected size: 130bp
  5. T4 (pBad, BBa_I13453) = 6ml + 1ml of dye = 7ml. Expected size: 130bp
  6. Ladder = 5 ml;

Purification (MiniPrep)

Part Description Plasmid Backbone Size, bp Colony Stock name Concentration, ng/ul
BBa_E0040
GFP
pSB1A2
720
T1
pRG.001
277.4
T2
pRG.002
233.3
BBa_I13453
pBad
pSB1A3
130
T3
pRG.003
246.4
T4
pRG.004
191.6
BBa_R0011
pLac
pSB1A2
55
T5
pRG.005
279.8
T6
pRG.006
215.4

07.07.2012

Transformation results

Successful transformation:

Start Culture for miniprep and glycerol

  • We pick 2 colonies from each plate.
  • Add it to 6 ml of LB + Ampicilline (6 tubes: T1, T2, T3, T4, T5, T6).
  • Overnight incubation.

Colony PCR of BBa_I13453 and BBa_E0040

Here, we run a PCR in order to run a verification gel afterwards (to make sure that the parts retreived from the registry are the right ones).

  1. Gently vortex and briefly centrifuge PCR Master Mix (2X) after thawing.
  2. Place a thin-walled PCR tube on ice and add the following components for each 50ul reaction:
  3. Chemical Volume
    PCR Master Mix (2X)
    25 ul
    Forward primer (VF2)
    2.5ul
    Reverse primer (VR)
    2.5ul
    Template DNA
    Put a loop
    Water, nuclease-free
    to 50ul
    Total volume:
    50ul


  4. Gently vortex the samples and spin down.
  5. When using a thermal cycler that doesn't contain a heated lid, overlay the reaction mixture with 25ul of mineral oil.
  6. Perform PCR using the recommended thermal cycling conditions outlined below:
  7. Step Temperature, °C Time Number of cycles
    Initial denaturation
    95
    10 min
    1
    Denaturation
    95
    30 s
    30
    Annealing
    50
    30 s
    Extension
    72
    1 min
    Final Extension
    72
    10 min
    1
    End
    8
    Forever
    1


  • Comments: LID = 100°C

06.07.2012

Biobrick Retrieval

Biobrick Retrieval Protocol was used. List of retrieval parts:

Part Description Location in a kit Plasmid Backbone
BBa_E0040
wild-type green fluorescent protein (GFP) derived from Jellyfish Aequeora victoria
2012 Kit Plate 1, well 14K
pSB1A2
BBa_I13453
pBad
2012 Kit Plate 1, well 1F
pSB1A3
BBa_R0011
pLac (positive control)
Antoine stock
pSB1A2

Heat Shock Transformation

  • Parts from the list above were transformed using Heat Shock Transformation protocol:
    20ml of competent NEB Turbo cells + 2ml of the plasmids.
  • The remaining plasmids (8ml) were stored at -20°C.
Personal tools