Team:Ionis Paris/Notebook/10 10 2016

October 10th 2016

PCR on G3

Objectives

The overall purpose is to amplify DNA fragment for further digestions and ligations in order to construct biobrick:

G3 : Gluc optimised —> isolation of Gluc from BBB to create BBG3

Materials

DNA fragments: BBB-4 (from mini prep 22/09/16)
Primers: Fw-Gluc (forward) and Rev-Gluc (reverse)

Protocol

PCR

1. Mix for 2 samples (Total volume of Mix : 96 µL), in an Eppendorf tube :

79.5 µL H2O

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

2 µL dNTP (200 µM final, NEB #N0447S)

2 µL Primer Fw-Gluc

2 µL Primer Rev-Gluc

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

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

48 µL Mix

2 µL of DNA fragment (BBB) or 2 µL H20 (Control)

—> Gently mix the reaction and short spin centrifugation

3. Set the following parameters for the PCR reaction :

G3 (617 bp):
Lid: 95°C
Initial denaturation: 95°C, 30 s
30 cycles of: 95°C, 30 s
50°C, 1 min
68°C, 37 s
Final extension: 68°C, 5 min
Hold: 4°C

Electrophoresis: for screening the PCR results

1% Agarose gel:

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

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

3. Add 5 µL of Gel Red 10,000 X (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 2 µL of Purple loading dye 6 X in 10 µL of sample.

3. Drop-off 10 µL of Purple ladder and 12 µL of each sample (G3: PCR product, CG3: PCR purified G3).



4. 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. Store the purified DNA at -20°C.

Results

Electrophoresis:

Expected results / Obtained results:



Interpretation

We obtain the desired strip for G3, as shown on the gel above the strips are closed to 617 bp.
It seems that G3 has been properly amplified.

Miniprep: on DH5⍺ transformed with BBG1

Objectives

Purification of BBG1 plasmid extracted from bacterial mini-cultures in order to have a stock of plasmid.

Materials

Mini-culture of bacteria transformed with BBG1 realized the 09/10 (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 4 miniprep were realized using the QIAprep® Spin Miniprep Kit (Qiagen, ref: 27104) and following 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.

12 tubes of BBG1 (3 per mini-cultures)

2. Store at -80°C.

Digestion: C4 and pSB1A2-C5; BB12 and BBX4; P12His and BB-B0015; G3 and pSB1C3

Objective

Double digestion of C4 by EcoRI and SpeI and of pSB1A2-C5 by EcoRI and XbaI for the subsequent ligation of C4 in pSB1A2-C5 to obtain C45.
Double digestion of BB12 by SpeI and PstI and of X4 by XbaI and PstI for the subsequent ligation of BBX4 in BB12 to obtain BBX5.
Double digestion of P12His by EcoRI and SpeI and of BB-B0015 by EcoRI and XbaI for the subsequent ligation of BB12his in term to obtain BBX3.
Double digestion of G3 by EcoRI and PstI and of pSB1C3-RFP by EcoRI and PstI for the subsequent ligation of G3 in pSB1C3 and creation of BBG3


Materials

Stock concentrations:

BBC5: ~ 150 ng/µL (from mini prep 02/10)
C4: ~ 50 ng/µL (from PCR 08/10)
BB12mut-4: ~ 250 ng/µL (from mini prep 07/09)
BBX4: ~ 100 ng/µL (from mini prep 07/10)
P12His: ~ 50 ng/µL (from PCR 08/10)
BB-B0015: ~ 150 ng/µL (from mini prep 08/10)
G3: ~ 50 ng/µL (from PCR 10/10)
pSB1C3-RFP 4: 94 ng/µL (from mini prep 19/07)


Protocol

Digestion:

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



NB: The digestion were done in 20 µL.

2. Short Spin Centrifugation

3. Incubation 1h at 37°C

4. Store at 4°C before purification

Electrophoresis for digested BBX4, pSB1C3-RFP:

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 3 µL of Gel Red 10,000X (0.3X 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 4 µL of Purple loading dye 6X in the 20 µL of sample.

3. Drop-off 10 µL of Purple ladder and 24 µL of sample.



4. Run at 90 V.

Gel purification for digested X4 and pSB1C3:

QIAquick Gel purification kit (Qiagen, 28704), according to the protocol given by the supplier (available here)

1. Excise the DNA fragment from the agarose gel. Gel slice Weigh = 0.342 g / 0.278 g

2. Add 3 volumes Buffer QG (1026 µL / 834 µL) to 1 volume of gel.

3. Incubate at 50°C for 10 min until the gel slice has completely dissolved. Vortex the tube every 2–3 min to help dissolve gel. The color of the mixture is yellow.

4. Add 1 gel volume isopropanol to the sample and mix.

5. Load 800 µL of each samples to the QIAquick column. Centrifuge for 1 min at 13,000 rpm and discard flow-through. Load the rest and spin again.

6. Add 500 µL Buffer QG. Centrifuge for 1 min at 13,000 rpm and discard flow-through.

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

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

9. Place QIAquick column into a clean 1.5 mL microcentrifuge tube.

10. Add 30 µL Buffer EB to the center of the QIAquick membrane, let stand for 1 min, and centrifuge for 1 min at 13,000 rpm.

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

PCR purification for digested P12His, BB-B0015, G3, C4, BBC5, BB12:

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

1. Add 5 volumes Buffer PB (100 µL) to 1 volume of the sample (20 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 PE. 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 30 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 the ligation.

Results

Electrophoresis:

Expected results / Obtained results:



Interpretation

The digestion of BBX4 was efficient, we get 2 strips at the end of the electrophoresis. The strip at 717 pb was the digested X4 that we purified for the subsequent ligation.
The digestion of pSB1C3-RFP was efficient, we get 2 strips at the end of the electrophoresis. The strip at 2070 pb was the digested pSB1C3 that we purified for the subsequent ligation.

Ligation: P12His into BB-B0015, G3 into pSB1C3, of C4 into BBC5 and of X4 into BB12

Objective

Ligation of P12His into BB-B0015 to create BBX3, of G3 into pSB1C3 to create BBG3, of C4 into BBC5 to create pSB1A2-C45 and of X4 into BB12 to create X5 for subsequent transformation and creation of a stock of transformed 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 :

BB-B0015 : 5 ng/µL (150 ng into 30 µL)
P12His : 2.5 ng/µL (75 ng dans 30 µL)
G3 : 3.3 ng/µL (100 ng into 30 µL)
pSB1C3 : 2.1 ng/µL (62 ng into 30 µL)
BBC5 : 5 ng/µL (150 ng into 30 µL)
C4 : 3.3 ng/µL (100 ng into 30 µL)
BB12 : 8.3 ng/µL (250 ng into 30 µL)
X4 : 0.8 ng/µL (25 ng into 30 µL )

Protocol

1. In the following order, add :



2. Mix by pipetting

3. Incubate for 1h at room temperature

Transformation: competent DH5⍺ cells with ligation products BBX3, BBX5, pSB1A2-C45 and BBG3

Objective

The objective is to transforme competent DH5⍺ cells with the ligation products BBX3, BBX5, pSB1A2-C45 and BBG3

Materials

6. 4 aliquot of 100 µL DH5⍺ competent cells (from the 20/09/16)

7. Plasmid DNA : Ligation product BBX3, BBX5, pSB1A2-C45 and BBG3

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

9. Petri dish LB+Amp: Cm concentration = 100 µg/mL

Materials

Experimental conditions achieved :



—> We need 8 LB+Cm plates+ 4 LB+Amp plates + 7 LB plates

Transformations protocol:

10. 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.

11. Add the ligation products to the cell mixtures.

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

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

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

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

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

17. Place at 37°C for 1h at 250 rpm.

18. Warm selection plates to 25°C.

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

20. Spread the corresponding volume onto each plate.

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

Results (obtain the 11/10)

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/Ampicilline resistance gene, present in pSB1C3/pSB1A2. A PCR colonie is necessary to check the size of the plasmid present in colonies, and therefore in order to know if bacteria incorpore the correct plasmid.

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