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

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Revision as of 12:29, 17 July 2012

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:

• The following protocol was used:

• After 10 min at 37°C (???)

Gel

From left to right: pRG.007 - pRG.018

Comments:

16.07.2012

15.07.2012

14.07.2012

13.07.2012

12.07.2012

Results of sequencing

Sequencing results for PLac and PBAD were consistent.

Start Culture of I13453, E0040 and positive control transformant

• Using two colonies that formed (colonies 1 and 2) in each plate (A, B and C)
• 6 mL of LB + Ampicilline
• Incubate over night

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

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

Colony PCR of I13453 and E0040

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

• We sent our tubes to be sequenced by gatc [1]

Biobrick Retrieval

• We retrieved various parts from the plates.
  1. BBa_E0240 (RBS BOO32 GFPmut3 TT):

     Kit 2012 Plate 1 well 12M

     Plasid: pSB1A2

  2. BBa_J09250 (pLacIq B0032 GFPmut3 TT)

     Kit 2012 Plate 2 well 9B

     Plasid: pSB2KB

  3. BBa_K117004 (pLacI B0030 GFPmut3 TT)

     Kit 2012 Plate 2 well 14J

     Plasid: pSB1A2

  4. BBa_I13540 (pBAD B0034 GFPmut3 TT)

     Kit 2007 Plate 2 well 17L

     Plasid: pSB1A3

  5. BBa_1746902 (pBAD B0034 GFPmut3 His_tagged)

     Kit 2012 Plate 3 well 16F

     Plasid: pSB1AK3

  6. BBa_J04450 (pLacI B0034 mRFP TT)

     Kit 2012 Plate 1 well 1G

     Plasid: pSB1A3

• We used Plac (R0011) as a positive control

• See Biobrick Retrieval Protocol here

Heat Shock Transformation

• We transformed 3*20mL of competent NEB Turbo cells with 2mL of the plasmids we just extracted from the plates (E0240, K117004, I746902, J04450, I13540) and our positive control R0011.
• The remaining plasmids (8mL) were stored at -20°C
• See Heat Shock Transformation protocol here

  Plate A: NEB turbo cells with E0240

  Plate B: NEB turbo cells with K117004

  Plate C: NEB turbo cells with I746902

  Plate D: NEB turbo cells with J04450

  Plate E: NEB turbo cells with I13540

  Plate F: NEB turbo cells with positive control

We let them grow over night at 37°c

08.07.2012

Glycerol Stock

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)

07.07.2012

List of parts T1-T6:

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 I13453 and 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.

Heat Shock Transformation

• We transformed 3*20mL of competent NEB Turbo cells with 2mL of the plasmids we just extracted from the plates (I13453, E0040) and our positive control R0011.
• The remaining plasmids (8mL) were stored at -20°C
• See Heat Shock Transformation protocol here

  Plate A: NEB turbo cells with I13453

  Plate B: NEB turbo cells with E0040

  Plate C: NEB turbo cells with positive control