Team:Ionis Paris/17 09 16
The overall purpose is to create PA (Pr - RBS - XylR - Term) and PB (Pu - RBS - Gaussia - Term) from our biosensor. DNA fragments : BB123mut-6 (from mini prep 07/09) Mix for 5 samples (Total volume of Mix: 230 µL), in an Eppendorf tube: 198.75 µL H2O 25 µL Buffer Taq (1 X final, NEB #B9014S) 5 µL dNTP (200 µM final, NEB #N0447S) 1.25 µL Taq polymerase (2.5 units / 50 µL PCR final, NEB #M0273S) Add in 4 PCR tubes, in the following order: 46 µL Mix 1 µL primer forward (A12 or BBB-F) 1 µL primer reverse (BBA-R or A13) 2 µL of DNA fragment (BB123mut) or 2 µL H20 (Controls A and B) —> Gently mix the reaction and perform a short spin centrifugation Set the following parameters for the PCR reaction : PA (2285 bp) Lid temperature: 95°C Initial denaturation : 95°C, 30s 30 cycles of : 95°C, 30 s 58°C, 60 s 68°C, 2 min 17 s Final extension : 68°C, 5 min Hold : 4°C PB (1037 bp) Lid temperature 95°C Initial denaturation : 95°C, 5 min 30 cycles of : 95°C, 30 s 58°C, 60 s 68°C, 1 min 02 s Final extension : 68°C, 5 min Hold : 4°C 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. 16 Mini-cultures of bacteria transformed with BB12mut (3, 4, 5, 7, 8, 9, 11, 12) and BB123mut (1, 2, 4, 5, 7, 10, 11, 13). 1st electrophoresis: Expected results / Obtained results 2nd electrophoresis: Expected results / Obtained results We obtain the desired strip for BB12mut, for all the colonies (n°1 to 14). As shown on the gel above, the strips are closed to 2.2 kb, which is the size of P1+P2. A sequencing is necessary to be sure of the obtained biobrick. We obtain the desired strip for BB123, for all the colonies except for n°7. As shown on the gel above, the strips are closed to 3.4 kb, which is the size of P1+P2+P3. A sequencing is necessary to be sure of the obtained biobrick.
PCR : on PA and PB
Objectives
Materials
Primers: A12 (forward) and BBA-R (reverse) for PA, and BBB-F (forward) and A13 (reverse) for PB.Protocol
PCR
Electrophoresis: for screening the PCR results
Mini-culture: bacteria transformed with BB12mut and BB123mut
Put the colony with satisfying PCR results from the plates divided into squares to a 50mL Falcon tube containing 5mL LB+Cm.Results
Interpretation
