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.

Digestion

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 : 

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

Cloning on fliL promoter

Protocol

We used the Taq polymerase to amplify this promoter.

• Preparation of the template :

Resuspension of 1 colony E.coli K12 strain MG 1655 in 100µl of water.

• Preparation of PCR mix :

1 µl O 124
1 µl O 125
1 µl template DNA
25 µL Quick Load Taq polymerase Mix
22 µL pure water

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

Name	Ligation	Description	Biobricks
MP144.1	L128.1	pFlgA-RFP D132 (FI) - D136 (FV)
MP144.2	L128.2
MP144.3	L128.3
MP144.4	L128.4
MP145.1	L129.1	pFlgB-RFP D133 (FI) - D136 (FV)
MP145.2	L129.2
MP145.6	L129.6
MP145.7	L129.7
MP146.1	L130.1	pFlhB-RFP D134 (FI) - D136 (FV)
MP146.2	L130.2
MP146.7	L130.7
MP146.8	L130.8

Glycerol Stocks

• Protocol (see #2)

• List of Stocks

Strain	Ligation	Description	Biobricks
S143.1	L128.1	pFlgA-RFP D132 (FI) - D136 (FV)
S143.2	L128.2
S143.3	L128.3
S143.4	L128.4
S144.1	L129.1	pFlgB-RFP D133 (FI) - D136 (FV)
S144.2	L129.2
S144.6	L129.6
S144.7	L129.7
S145.1	L130.1	pFlhB-RFP D134 (FI) - D136 (FV)
S145.2	L130.2
S145.7	L130.7
S145.8	L130.8

Results of the transformations we did yesterday

PCR screening of the transformations we did yesterday

Concentration of the MiniPreps

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