Team:Ionis Paris/25 07 16
NB: BB1 and BB2 are the ligation products of P1 + pSB1C3 and P2 + pSB1C3. The overall purpose is to check if the bacteria obtain from the transformation with BB1 and BB2 are containing the good genetic construction. Bacteria tansformed with BB1 and BB2 (made on 21/07/16) 1. 1 Mix for 18 samples of BB1 (Total volume Mix : 900 µL) in a Eppendorf tube : 747 µL H2O 90 µL Buffer Taq (1 X final, NEB #B9014S) 18 µL Primer A12 (1µM final) 18 µL Primer A13 (1µM final) 18 µL dNTP (200µM final, NEB #N0447S) 9 µL Taq polymerase (2.5units/50 µL PCR final, NEB #M0273S 2. 1 Mix for 7 samples of BB2 (Total volume Mix : 350 µL) in a Eppendorf tube : 290.5 µL H2O 35 µL Buffer Taq (1 X final, NEB #B9014S) 7 µL Primer VF (1 µM final) 7 µL Primer VR (1 µM final) 7 µL dNTP (200µM final, NEB #N0447S) 3.5 µL Taq polymerase (2.5 units / 50 µL PCR final, NEB #M0273S) 3. Add in 21 PCR tubes, in the respected order: 50 µL Mix One colony from the different petri dish (pick one colony, put some on a new, divided into squares petri dish and put the remaining bacteria into the PCR mix) Gently mix the reaction 4. Short spin centrifugation 5. Set the following parameters for the PCR reaction : Part 1 (532 bp) Lid température 95°C Initial denaturation : 95°C, 5 min 30 cycles of : 95°C, 30 s 58°C, 1 min 68°C, 30 s Final extension : 68°C, 5 min Hold : 4°C Part 2 (1,785 bp) Lid température 95°C Initial denaturation : 95°C, 5 min 30 cycles of : 95°C, 30 s 58°C, 1 min 68°C, 1 min 42 s Final extension : 68°C, 5 min Hold : 4°C 1% Agarose gel: Put 1g of agarose low melting point + 100 mL of TAE 1 X 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 10,000X (0.5 X final) Flow the gel and place the combs Wait until it is solidified. Remove slowly the combs. Drop-off: 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 80V 11 Mini-cultures of bacteria transformed with BB1 (colonies 1,2,3,7,8,9,10,11,12,14,15): < font color = ”46BB0A”> 1st electrophoresis: Expected results / Obtained results: < font color = ”46BB0A”> 2nd electrophoresis: Expected results / Obtained results: We obtain the desired strip for BB1, as shown on the gel above the strips are close to 0.5 kb, which is the size of P1. A sequencing is necessary to be sure of the obtained biobrick. The objective is to test the competency of the DH5⍺ cells done on the 23/07/16 with the stock of pSB1C3-RFP plasmid. Plasmid DNA : « pSB1C3-RFP 1 » at 85.151 ng/µL DH5⍺ competent cells (23/07/16) Petri dish LB+Cm: Cm concentration = 25 µg/mL We need 5 LB+Cm plates + 2 LB plates Thaw 1 tube of DH5α competent cells on ice for 10 min. Mix gently and carefully pipette 50 µL of cells into 2 transformation tubes on ice. Add 1 µL (85.151 ng) of plasmid DNA to the cell mixture. Carefully flick the tube 4-5 times to mix cells and DNA. Do not vortex. Place the mixture on ice for 30 min. Do not mix. Heat shock at exactly 42°C for 30 s. Do not mix. Place on ice for 5 min. Do not mix. Pipette 950 µL of room temperature SOC into the mixture. Place at 37°C for 60 min at 250 rpm. Warm selection plates to 25°C. Mix the cells thoroughly by flicking the tube and inverting. Spread 100 µL of each dilution (100%,10%,1%) onto a selection plate. Pellet the remaining cells (except for the control), discard mostly all the supernatant and resuspend the remaining cells in 100 µL of LB. Spread 100 µL onto a selection plate. Incubate all the plates O/N at 37°C. Expected results : Some colonies on the petri dishes LB+Cm plated with 1% of transformed bacteria, more with 10%, a lot with 100% and a bacterial lawn with the pellet. Obtained results: We obtain the expected results. The transformation worked, we obtained satisfying results. Colonies contain a plasmid with the Chloramphenicol resistance gene, present in pSB1C3. The DH5α cells are competent.
PCR colony: on colonies transformed by BB1 and BB2
Objectives
Materials
Primers: A12 (forward) and A13 (reverse). Protocol
PCR
Electrophoresis for screening the PCR results
Mini-culture: bacteria transformed with BB1
Put the colony with satisfying PCR results from the plates divided into squares to a 50 mL Falcon tube containing 5 mL LB+CHL. Results
Electrophoresis for screening the PCR results
Interpretation
However, concerning P2, some strip can be seen at 100 pb, meaning that P2 has not been ligated into pSB1C3, our bacteria have integrated only the plasmid pSB1C3 that has been closed up on itself.
Transformation efficiency test : Competent DH5⍺ cells with pSB1C3-RFP plasmid
Objectives
Materials
Protocol
Experimental conditions:
Results
A bacterial lawn is expected on the LB petri dishes (+ control).
No colony is expected on the LB+Cm petri dishes plated with no plasmid (- control).
All obtained colonies are expected to be red because of the RFP gene that codes for a red fluorescent protein.
Interpretation
