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

23.07.2012

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 of PCR BBa_I13453 (pBad) and BBa_R0011 (pLac)

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gel_23_07_2012.gif

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:

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

3. Gently mix the samples.
4. Perform PCR using the recommended thermal cycling conditions outlined below:

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:

• BBa_K098991
• BBa_K093012
• BBa_B0032

Unsuccessful transformation:

• BBa_R0051

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:

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




3. Gently vortex the samples and spin down.
4. When using a thermal cycler that doesn't contain a heated lid, overlay the reaction mixture with 25ul of mineral oil.
5. Perform PCR using the recommended thermal cycling conditions outlined below:

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.

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

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

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 want to sequence pBAD (tubes pRG003 and pRG004) and pLac (tube pRG005)

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

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

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:

• BBa_E0040
• BBa_I13453
• BBa_R0011

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

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:

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




3. Gently vortex the samples and spin down.
4. When using a thermal cycler that doesn't contain a heated lid, overlay the reaction mixture with 25ul of mineral oil.
5. Perform PCR using the recommended thermal cycling conditions outlined below:

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.