Team:Paris/August 12

Digestion and ligation of the PCR we did yesterday
Yesterday we amplified flhD, flhC, flhDC with its promoter and E040 RBS+ Before the digestion, we have to determine the DNA concentration of the templates.

Measurement of DNA concentration
We used the biophotometer. We put 5 µL of template DNA in 95 µL of water. The Blank was made with 5 µL of EB Buffer (the one that was used for the elution of the DNA) in 95 µL of water.

Protocol

 * X µL of Template DNA
 * Buffer (n°2) 10X : 3µL
 * BSA 100X : 0.3µL
 * Pure water qsp 30 µL
 * 1 µL of each enzyme


 * Incubate during about 3h at 37°C, then 20 minutes at 65°C (to inactivate the enzymes).
 * Exclusively for D 144 : 2 µL of Antarctic Phosphatase was added, 30 min at 37°C then 5 min at 65°C.

Results of the digestion
For the digestion after PCR, an electrophoresis is useless : we can not separate DNA fragments that have 10 bp of difference. Exclusively for D 145 a gel extraction was made because there was two fragments, the standard protocol was used (the picture is missing because we hadn't the USB key). The DNA purification after gel extraction was done according the standard protocol.

Ligation
REMARKS : forgot to do the autoligation control, which is a key step in the evaluation of the ligation process.

Protocol
We used the Taq polymerase to amplify this promoter.

Resuspension of 1 colony E.coli K12 strain MG 1655 in 100µl of water.
 * Preparation of the template :

1 µl O 124 1 µl O 125 1 µl template DNA 25 µL Quick Load Taq polymerase Mix 22 µL pure water
 * Preparation of PCR mix :

Result
We did an electrophoresis to check if our amplicon has the right size. Actually, the electrophoresis did not show anything, not even the DNA ladder. We decided to do again the electrophoresis tomorrow morning.

Minipreps: Plasmid extraction

 * Protocol (see # 3)  Experiments done by QIAcube


 * List of Minipreps

Glycerol Stocks

 * Protocol (see #2)


 * List of Stocks

New PCR screening with the right primers
Transformants with pFlgA, pFlgB and pFlhB cloned into J61002 are analysed by PCR but this time with the right primers: VF2 (O18) and VR (O19).
 * template: bacteria from glycerol stock
 * positive control: J61002 (with ptet-mRFP)
 * negative control: no template

Electrophoresis

 * 1% agarose gel
 * 10 µL of each sample loaded