October 18th 2016

Colony PCR: on colonies transformed by BB2mut, BBX2, BBC4 and BBG2

NB: BB2mut is the ligation product of pSB1C3 + P2mut, BBX2 is the ligation product of pSB1C3 + X2, BBC4 is the ligation product of pSB1C3 + C4 and BBG2 is the ligation product of pSB1C3 + G2.

Objectives

PThe overall purpose is to check if the bacteria obtain from the transformations with BB2mut, BBX2, BBC4 and BBG2 contain the good genetic constructions.

Materials

Bacteria tansformed with BB2mut, BBX2, BBC4 and BBG2 (made on 17/10/16).
Primers: A12 (forward) and A13 (reverse).

Protocol

PCR

1. 1 Mix for 15 samples (Total volume of each Mix : 750 µL), in an Eppendorf tube :

360 µL H2O

7.5 µL Primer A12 (0.5 µM final)

7.5 µL Primer A13 (0.5 µM final)

375 µL Q5 High-Fidelity 2X Master Mix (NEB #M0492S)

2. Add in 42 PCR tubes, in the respected order:

50 µL Mix

One colony from the different petri dishes (pick one colony, put some on a new plate, divided into squares, and put the remaining bacteria into the PCR mix)

—> Gently mix the reaction

3. Short spin centrifugation

4. Set the following parameters for the PCR reaction :

P2mut (1,747 bp)
Lid température: 98°C
Initial denaturation: 98°C, 5 min
25 cycles of: 98°C, 10 s
64°C, 30 s
72°C, 52 s
Final extension: 72°C, 2 min
Hold: 4°C

X2 (1,794 bp)
Lid température: 98°C
Initial denaturation: 98°C, 5 min
25 cycles of: 98°C, 10 s
64°C, 30 s
72°C, 52 s
Final extension: 72°C, 2 min
Hold: 4°C

C4 (402 bp)
Lid température: 98°C
Initial denaturation: 98°C, 5 min
25 cycles of: 98°C, 10 s
64°C, 30 s
72°C, 12 s
Final extension: 72°C, 2 min
Hold: 4°C

G2 (707 bp)
Lid température: 98°C
Initial denaturation: 98°C, 5 min
25 cycles of: 98°C, 10 s
64°C, 30 s
72°C, 21 s
Final extension: 72°C, 2 min
Hold: 4°C

Electrophoresis: for screening the PCR results

1% Agarose gel:

Put 1 g of agarose + 100 mL of TAE 1X in a bottle of 500 mL
Mix and heat it 2 min 30 s in the microwaves. Wait the cooling of the bottle until it is tepid.
Add 5 µL of Gel Red 10,000 X (0.5 X final)
Flow the gel and place the combs
Wait until it is solidified. Remove slowly the combs.

Drop-off:

Short Speed centrifugation of samples.
Addition of 2 µL of Purple loading dye 6 X in 10 µL of sample.
Drop-off 10 µL of Purple ladder and 12 µL of each samples.

Run at 90 V.

Mini-culture: bacteria transformed with BBA and BBB

8 Mini-cultures of bacteria transformed with BBC4 (2, 4, 6), BBG2 (4), BBP2mut (1, 2, 4):
Put the colony with satisfying PCR results from the plates divided into squares to a 50 mL Falcon tube containing 5 mL LB+Cm.

Results

1st electrophoresis: Expected results / Obtained results :

2nd electrophoresis: Expected results / Obtained results :

Interpretation

We obtain desired strips for BBP2mut (n°1,2,4), BBC4 (n°2,4,6) and BBG2 (n°4). As shown on the gel above, the strips are closed, respectively, to 1,747 bp, 402 bp and 707 bp, which are the sizes of P2mut, C4, G2. A sequencing is necessary to be sure of the obtained biobrick.

Concerning BBX2, we obtained 2 strips at the size of mRFP device present in pSB1C3-RFP (1069 bp). The absence of correct clones is due to the lake of PstI restriction site on the X2 part and therefore the impossibility to insert it in pSB1C3 with the made digestion. The X2 DNA fragment does not contain PstI restriction site because it that has been amplified thanks to 2 primers, a forward primer that has the Prefix in overhang and a reverse primer that has a reduced version of the Suffix without PstI restriction site in overhang. This reverse primer was too long to contain the whole suffixe, due to its overhang containing also the His-tag. It is already composed of 60 bp, the maximum length to synthesised a primer.
Therefore, we have to digest X2 and pSB1C3-RFP by EcoRI and SpeI in order to insert X2 in pSB1C3 to obtain BBX2.