IGEM:Paris Bettencourt 2012/Notebook/RE group/Lab notes: Difference between revisions
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==14.08.2012== | ==14.08.2012== | ||
==Verification gel== | |||
*1% agar gel, 1kb ladder, 48 reactions + 1 neg control | |||
*expected sizes: | |||
*#pBAD+RBS+IsceI+PSB3C5 (pRG106): 1151 | |||
*#pBAD+RBS+GFP+PSB3C5 (pRG107): 1294 | |||
*#pBAD+RBS+RFP+PSB3C5 (pRG108): 1118 | |||
*#pLac+RBS+IsceI+PSB3C5 (pRG109): 1076 | |||
*#pLac+RBS+GFP+PSB3C5 (pRG110): 1219 | |||
*#pLac+RBS+RFP+PSB3C5 (pRG111): 1043 | |||
*#pRHA+RBS+IsceI+PSB3C5 (pRG112): 1143 | |||
*#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== | ==13.08.2012== | ||
Revision as of 08:35, 24 August 2012
14.08.2012
Verification gel
- 1% agar gel, 1kb ladder, 48 reactions + 1 neg control
- expected sizes:
- pBAD+RBS+IsceI+PSB3C5 (pRG106): 1151
- pBAD+RBS+GFP+PSB3C5 (pRG107): 1294
- pBAD+RBS+RFP+PSB3C5 (pRG108): 1118
- pLac+RBS+IsceI+PSB3C5 (pRG109): 1076
- pLac+RBS+GFP+PSB3C5 (pRG110): 1219
- pLac+RBS+RFP+PSB3C5 (pRG111): 1043
- pRHA+RBS+IsceI+PSB3C5 (pRG112): 1143
- 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
- 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
- Wait while OD = 1.25
- Wash cells and add 10 ml LB without antibiotic.
- 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
- Overnight grow.
- Mesure OD (OD = 2.95 for both tubes)
- Dilute it (x3000)
- Plate 50 ul and 100 ul from each tube on 2 kinds of plate:
- Amp;
- Amp + Cm;
- Wait overnight to get results
Run a gel (Colony PCR results)
 |
 |
 |
 |
- 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
- First gel:
The size is right for all parts.
07.08.2012
Colony PCR
- The standard protocol for PCR with Taq polymerase was used
- 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.062 |
pRG.066; pRG.067; pRG.068; | ||
pRG.070 |
pRG.074; pRG.075; pRG.076; | ||
pRG.078 |
pRG.082; pRG.083; pRG.084; | ||
06.08.2012
Run a gel (Ligation results)
- 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 | |
- Digestion time: 75 min.
- Digestion temperature: 37°C.
- Shaking: 400 rpm
Run a gel (Digestion)
- 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)
- Digestion of pRG.055 (RBS + I-SceI + 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
- Digestion of pSB3C5
- First gel:
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;
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
- The standard protocol for PCR with Taq polymerase was used
- 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 | |
pRG.054 |
pRG.056 | |
pRG.058 |
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:
- Digestion time: 60 min.
- Digestion temperature: 37°C.
Digestion of Vectors
| Reagent | Volume | |
|---|---|---|
| RFP | RBS + pSB1A2 | |
- Digestion time: 75 min.
- Digestion temperature: 37°C.
Run a gel (Digestion)
 |
 |
- 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
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)
- The standard protocol for PCR with Taq polymerase was used
- 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)
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
- Biobrick retrieval and transformation was done by following the standard protocols:
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:
- 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 |
|---|---|---|---|
pLac digestion
- Digestion plasmids from miniprep stock:
- pRG.035 (114 ng/uL)
- pRG.037 (152 ug/uL)
- pRG.039 (143 ng/uL)
- Pipetting instructions:
- 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:
- pRG.009, pRG.010: pLacI + RBS (B0030) + GFP + TT + pSB1A2;
- pRG.013, pRG.014: pLacI + RBS (B0034) + RFP + T + pSB1A3;
- pRG.011, pRG.012: pBad + RBS (B0034) + GFP + pSB1AK3;
- pRG.015, pRG.016: pBad/araC + RBS (B0034) + GFP + TT + pSB1A3;
- 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
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:
- We ligate pBAD with RBS and pBAD with RBS/GFP
- 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
- Gently vortex and briefly centrifuge PCR Master Mix (2X) after thawing.
- Place a thin-walled PCR tube on ice and add the following components for each 50ul reaction:
- Gently vortex the samples and spin down.
- When using a thermal cycler that doesn't contain a heated lid, overlay the reaction mixture with 25ul of mineral oil.
- Perform PCR using the recommended thermal cycling conditions outlined below:
| Chemical | Volume |
|---|---|
| Step | Temperature, °C | Time | Number of cycles |
|---|---|---|---|
- Comments: LID = 100°C
Gel Results
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)
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.
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 |
- Gently mix PCR Master Mix (2X) after thawing.
- Place a thin-walled PCR tube on ice and add the following components for each 50ul reaction:
- Gently mix the samples.
- Perform PCR using the recommended thermal cycling conditions outlined below:
| Chemical | Volume |
|---|---|
| Step | Temperature, °C | Time | Number of cycles |
|---|---|---|---|
- Comments: LID = 100°C
22.07.2012
Glycerol Stock
| Part | Description | Plasmid Backbone | Size, bp | Colony | Stock name |
|---|---|---|---|---|---|
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
- Gently vortex and briefly centrifuge PCR Master Mix (2X) after thawing.
- Place a thin-walled PCR tube on ice and add the following components for each 50ul reaction:
- Gently vortex the samples and spin down.
- When using a thermal cycler that doesn't contain a heated lid, overlay the reaction mixture with 25ul of mineral oil.
- Perform PCR using the recommended thermal cycling conditions outlined below:
| Chemical | Volume |
|---|---|
| Step | Temperature, °C | Time | Number of cycles |
|---|---|---|---|
- Comments: LID = 100°C
20.07.2012
Biobrick Retrieval Protocol was used. List of retrieval parts:
| Part | Description | Location in a kit | Plasmid Backbone |
|---|---|---|---|
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 |
|---|---|---|---|---|
- The following protocol was used:
| Chemical | Volume |
|---|---|
- After 10 min at 37°C (???)
Gel
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:
- Ladder = 5 ml;
- pRG.007 (GFP, BBa_E0240) = 5ml + 1ml of dye = 6ml. Expected size: 720bp
- pRG.008 (GFP, BBa_E0240) = 5ml + 1ml of dye = 6ml. Expected size: 720bp
- pRG.009 (pBad, BBa_K117004) = 5ml + 1ml of dye = 6ml. Expected size: 130bp
- pRG.010 (pBad, BBa_K117004) = 5ml + 1ml of dye = 6ml. Expected size: 130bp
- pRG.011 (pBad, BBa_I746902) = 5ml + 1ml of dye = 6ml. Expected size: 130bp
- pRG.012 (pBad, BBa_I746902) = 5ml + 1ml of dye = 6ml. Expected size: 130bp
- pRG.013 (pBad, BBa_J04450) = 5ml + 1ml of dye = 6ml. Expected size: 130bp
- pRG.014 (pBad, BBa_J04450) = 5ml + 1ml of dye = 6ml. Expected size: 130bp
- pRG.015 (pBad, BBa_I13540) = 5ml + 1ml of dye = 6ml. Expected size: 130bp
- pRG.016 (pBad, BBa_I13540) = 5ml + 1ml of dye = 6ml. Expected size: 130bp
- pRG.017 (pBad, BBa_R0011) = 5ml + 1ml of dye = 6ml. Expected size: 130bp
- pRG.017 (pBad, BBa_R0011) = 5ml + 1ml of dye = 6ml. Expected size: 130bp
- Ladder = 5 ml;
13.07.2012
Purification (MiniPrep)
| Part | Description | Plasmid Backbone | Size, bp | Colony | Stock name | Concentration, ng/ul |
|---|---|---|---|---|---|---|
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:
- 750uL of solution from the culture tube
- 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
- Mix 50mL of BET (0.5%) with 0.5g of agarose
- Put in the microwave 1.30min and then an additional 20s (till the solution is transparent)
- Wait a little bit till it cools down (no more steam)
- Pull in a big plate (to make a big size gel)
- Wait 30min
- For each PCR tube: Mix 1uL of die with 5uL of solution from the PCR tube
- Put this mix in gel wells
- Wait for migration (approx 30min). Voltage: 100V
- Put the gel in an ethidium bromide bath for 10min
- Rince in H2O destain for 5min
- Take picture
From left to right, we've put the following parts in well:
- Ladder (1kb)
- E0240 colony 1 (pcr tube 1)
- E02410 colony 2 (pcr tube 2)
- Ladder 1kB
- K117004 colony 1 (pcr tube 3)
- K117004 colony 2 (pcr tube 4)
- I746902 colony 1 (pcr tube 5)
- I746902 colony 2 (pcr tube 6)
- J04450 colony 1 (pcr tube 7)
- J04450 colony 2 (pcr tube 8)
- I13540 colony 1 (pcr tube 9)
- I13540 colony 2 (pcr tube 10)
- Control+ R0011 colony 1 (pcr tube 11)
- Control+ R0011 colony 2 (pcr tube 12)
- K117004 colony 1 (pcr tube 3)
- 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.
- 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
- The primers we used are VR and VF2 at a concentration of 10uM
| 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 |
|---|---|---|---|
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 |
|---|---|---|---|---|---|
Gel
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:
- Ladder = 5 ml;
- T1 (GFP, BBa_E0040) = 6ml + 1ml of dye = 7ml. Expected size: 720bp
- T2 (GFP, BBa_E0040) = 6ml + 1ml of dye = 7ml. Expected size: 720bp
- T3 (pBad, BBa_I13453) = 6ml + 1ml of dye = 7ml. Expected size: 130bp
- T4 (pBad, BBa_I13453) = 6ml + 1ml of dye = 7ml. Expected size: 130bp
- Ladder = 5 ml;
Purification (MiniPrep)
| Part | Description | Plasmid Backbone | Size, bp | Colony | Stock name | Concentration, ng/ul |
|---|---|---|---|---|---|---|
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).
- Gently vortex and briefly centrifuge PCR Master Mix (2X) after thawing.
- Place a thin-walled PCR tube on ice and add the following components for each 50ul reaction:
- Gently vortex the samples and spin down.
- When using a thermal cycler that doesn't contain a heated lid, overlay the reaction mixture with 25ul of mineral oil.
- Perform PCR using the recommended thermal cycling conditions outlined below:
| Chemical | Volume |
|---|---|
| Step | Temperature, °C | Time | Number of cycles |
|---|---|---|---|
- 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 |
|---|---|---|---|
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.
