Team:Ionis Paris/08 10 2016

October 8th 2016

PCR on C4, X3, G2

Objectives

The overall purpose is to amplify different DNA fragments for further digestions and ligations in order to construct biobricks:

• C4 : Pu + RBS —> isolation of Pu-RBS from BBB to create BBC45 (Pu-RBS-GFP-term)

• X3 : XylR - His tag —> addition of a His tag to XylR using BB12mut

• G2 : Gluc + term —> isolation of Gluc-term from BBB to create BBG1 (Pr-RBS-Gluc-Term

  )

Materials

DNA fragments:
- BBB-4 (from mini prep 22/09)
- BB12mut-3 (from mini prep 07/09)
Primers:
- A12 (forward) and Rev-C4 (reverse) for C4
- Fw-G2 (forward) and A13 (reverse) for G2
- A12 (forward) and Rev-XylHis (reverse) for BB12his

Protocol

PCR:

1. Mix for 10 samples (Total volume of Mix : 460 µL), in an Eppendorf tube :

   • 397.5 µL H2O

   • 50 µL Buffer Taq (1 X final, NEB #B9014S)

   • 10 µL dNTP (200 µM final, NEB #N0447S)

   • 2.5 µL Taq polymerase (2.5 units / 50 µL PCR final, NEB #M0273S)

2. Add in 10 PCR tubes, in the respected order:

   • 46 µL Mix

   • 1 µL Primer Forward (A12 or Fw-G2)

   • 1 µL Primer Reverse (Rev-C4, A13 or Rev-XylHis)

   • 2 µL of DNA fragment (BBB or BB12mut) or 2 µL H20 (Controls 1, 2, 3)

3. —> Gently mix the reaction and short spin centrifugation

4. Set the following parameters for the PCR reaction :

   • - C4 (402 bp):

   • Lid temperature 95°C

   • Initial denaturation : 95°C, 30 s

   • 30 cycles of :/li>

   • • 95°C, 30 s

     • 56°C, 1 min

     • 68°C, 22 s

     • Final extension : 68°C, 5 min

   • Hold : 4°C

• - G2 (707 bp):

• Lid temperature 95°C

• Initial denaturation : 95°C, 30 s

• 30 cycles of :/li>

• • 95°C, 30 s

  • 58°C, 1 min

  • 68°C, 41s

  • Final extension : 68°C, 5 min

• Hold : 4°C

• - P12his (2183 bp):

• Lid temperature 95°C

• Initial denaturation : 95°C, 30 s

• 30 cycles of :/li>

• • 95°C, 30 s

  • 62°C, 1 min

  • 68°C, 41s

  • Final extension : 68°C, 2min 11s

• Hold : 4°C

Electrophoresis: for screening the PCR results :

1% Agarose gel:

1. Put 1 g of agarose low melting point + 100 mL of TAE 1X in a bottle of 500 mL.

2. Mix and heat it 2min 30s in the microwaves. Wait the cooling of the bottle until it is tepid.

3. Add 5 µL of Gel Red 10,000X (0.5 X final).

4. Flow the gel and place the combs.

5. Wait until it is solidified. Remove slowly the combs.

Drop-off:

1. Short Speed centrifugation of samples.

2. Addition of 1 µL of Purple loading dye 6X in 5 µL of each sample.

3. Drop-off 10 µL of Purple ladder and 6 µL of each samples.

Plan:



Run at 90 V.

PCR Purification:

QIAquick PCR purification kit (Qiagen, 28106), according to the protocol given by the supplier (available here)

1. Add 5 volumes Buffer PB (250 µL) to 1 volume of the PCR reaction (50 µL) and mix. The color of the mixture is yellow.

2. Load the sample to the QIAquick column. Centrifuge for 1 min at 13,000 rpm and discard flow-through.

3. Add 750 µL Buffer. Centrifuge for 1 min at 13,000 rpm and discard flow-through.

4. Centrifuge once more for 1 min at 13,000 rpm.

5. Place each QIAquick column in a clean 1.5 mL microcentrifuge tube.

6. Add 50 µL Buffer EB to the center of the QIAquick membrane, let stand for 1 min, and centrifuge for 1 min at 13,000 rpm .

7. Calculate the quantity of DNA with the Nanodrop.

8. Store the purified DNA at -20°C.

Results

< font color = ” 46BB0A”> Expected Results :



< font color = ” 46BB0A”> Obtained Results :



Interpretation

We obtained the desired strip for G2, C4 and P12his, as shown on the gel above the strips are closed to 707 bp, 402 bp and 2,183 bp.
It seems that G2, C4 and P12his have been properly amplified.

Miniprep: on DH5⍺ transformed with BB-B0015

Objectives

Amplification of BB-B0015 plasmid extracted from bacterial mini-cultures in order to have a stock of plasmid and to construct BBX1 and BBX3.

Materials

2 Mini-cultures of bacteria transformed with BB-B0015 realized the 07/10 (put a colony with satisfying PCR results in 5 mL LB+Cm into a 50 mL Falcon tube).
From those mini-cultures, take 500 µL to realize a glycerol stock of tranformed bacteria. The 4.5 mL remaining will serve for the miniprep.

Protocol

The miniprep were realized using the QIAprep® Spin Miniprep Kit (Qiagen, ref: 27104) , according to the protocol given by the supplier (available here).

-Miniprep:

1. Divide each 4.5 mL bacterial O/N mini-cultures into 4 Eppendorf tubes and centrifuge all those tubes at 9,000 rpm for 3 min at room temperature. Discard the supernatant.

2. Resuspend the pellet in 62.5 μL Buffer P1 and pool the 4 Eppendorf tubes into a unique tube.

3. Add 250 μL Buffer P2 and mix by inverting the tube 6 times. The solution turns blue.

4. Add 350 μL Buffer N3 and mix by inverting the tube 6 times. The solution turns colorless.

5. Centrifuge for 10 min at 13,000 rpm.

6. Load 800 μL supernatant from step 5 to the QIAprep 2.0 spin column. Centrifuge for 1 min and discard the flow-through.

7. Add 500 µL Buffer PB. Centrifuge for 1 min at 13,000 rpm and discard the flow-through.

8. Add 750 µL Buffer PE. Centrifuge for 1 min at 13,000 rpm and discard the flow-through

9. Centrifuge once more for 1 min at 13,000 rpm.

10. Place the QIAprep 2.0 spin column in a clean 1.5 mL microcentrifuge tube.

11. Add 50 μL Buffer EB to the center of the QIAprep 2.0 spin column, let stand for 1 min, and centrifuge for 1 min at 13,000 rpm.

12. Store the purified DNA at -20°C.

Bacteria storage:

1. Add 100 µL of glycerol 50% to 100 µL of transformed bacteria in clean microcentrifuge 1.5 mL Eppendorf.

   • 6 tubes of BB-B0015 (3 per mini-culture)

2. Store at -80°C

Digestion: G2 and BB1

Objectives

Double digestion of G2 by XbaI and PstI, and of BB1 by SpeI and PstI for the subsequent ligation of G2 in BB1 to obtain BBG1.

Materials

- Stock concentrations:

BB1-2: 97.3 ng/µL (from miniprep 26/07)
G2: ~50 ng/µL (from PCR 08/10)

- Quantity of DNA required for the ligation of G2 into BB1:

BB1: Digestion of 250 ng (50 ng needed)
G2: Digestion of 50 ng (40.38 ng needed for the ratio 3:1)

Protocol

Digestion:

1. In a 1.5 mL Eppendorf tube, adding in the respected order (bigger volume first and enzyme last) :



NB: The digestions were done in 20 µL

2. Short Spin centrifugation

3. Incubation 1h at 37°C

4. Store at 4°C before purification

PCR purification for digested BB1, G2:

QIAquick PCR purification kit (Qiagen, 28106), according to the protocol given by the supplier (available here)

1. Add 5 volumes Buffer PB (250 µL) to 1 volume of the PCR reaction (50 µL) and mix. The color of the mixture is yellow.

2. Load the sample to the QIAquick column. Centrifuge for 1 min at 13,000 rpm and discard flow-through.

3. Add 750 µL Buffer. Centrifuge for 1 min at 13,000 rpm and discard flow-through.

4. Centrifuge once more for 1 min at 13,000 rpm.

5. Place each QIAquick column in a clean 1.5 mL microcentrifuge tube.

6. Add 50 µL Buffer EB to the center of the QIAquick membrane, let stand for 1 min, and centrifuge for 1 min at 13,000 rpm .

7. Store the purified DNA at 4°C before ligation.

Ligation: G2 into BB1

Objectives

Ligation of G2 into BB1 to obtain BBG1, for subsequent transformation and creation of a stock of bacteria.
The molar ratios for the ligation were calculated using NEB BioCalculator (available here)

Materials

- Concentrations of the different components after digestion and PCR purification :

BB1: 8.33 ng/µL (250 ng / 30 µL)
G2: 1.6 ng/µL (50 ng / 30 µL)

Protocol

1. In the following order, add :



2. Mix by pipetting

3. Incubate at Room Temperature

Transformation: competent DH5⍺ cells with ligation product BBG1

Objectives

The objective is to transforme competent DH5⍺ cells with the ligation product BBG1

Materials

3. 2 aliquot of 100 µL DH5⍺ competent cells (from the 20/09/16)

4. Plasmid DNA : Ligation product BBG1

5. Petri dish LB+Cm: Cm concentration = 25 µg/mL

Protocol

Experimental conditions achieved:



We need 4 LB+Cm plates + 3 LB plates

Transformation protocol:

1. Thaw tubes of DH5⍺ competent cells on ice for 10 min. Mix gently and carefully pipette 50 µL of cells into the 4 transformation tubes on ice.

2. Add the 40 µL plasmid DNA to the cell mixture.

3. Carefully flick the tubes 4-5 times to mix cells and DNA. Do not vortex.

4. Place on ice for 30 min. Do not mix.

5. Heat shock at exactly 42°C for 45 s. Do not mix.

6. Place on ice for 5 min. Do not mix.

7. Pipette 250 µL of room temperature SOC into the mixture.

8. Place at 37°C for 1h at 250 rpm.

9. Warm selection plates to 25°C.

10. Mix the cells thoroughly by flicking the tubes and inverting.

11. Spread the corresponding volume onto each plate.

12. Incubate all the plates O/N at 37°C.

Results (obtained on the 09/10)

Expected Results

Some colonies on the petri dishes LB+Cm plated with 50 µL of bacteria transformed with the different ligation products and more on the petri dishes LB+Cm plated with 200 µL of bacteria.
A bacterial lawn on the LB petri dishes without antibiotic.
No colonies on the LB+Cm petri dish plated with bacteria transformed with no plasmid (- control).

Obtained Results

We obtained expected results.

Interpretation

The transformation worked. Colonies contain a plasmid with the Chloramphenicol resistance gene, present in pSB1C3. However, this resistance can be due to the plamid BB1. It is possible that BB1 has closed up on itself. A PCR colonie is necessary to check the size of the plasmid present in colonies, and therefore in order to know if bacteria incorporated the correct plasmid.



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