Team:Paris/August 6

Assay to clone different flagella gene promotors into the J61002 plasmid

We have to digest (by EcoRI and SpeI) the PCR products of yesterday (amplification of pFlgA (MP124), pFlgB (MP125), pFlhB (MP126) and pFlhDC (MP127) in order to clone them into the J61002 plasmid (that we must extract and then digest by EcoRI and SpeI).

Plasmid extraction

The J61002 plasmid was extracted from overnight bacteria culture using the QIAspin Miniprep Kit (QIAGEN).

• Number of this plasmid extraction : MP123
• Carried out 3 times (3 tubes)

Assay to quantify DNA

In order to quantify the DNA contained either in the Miniprep product of MP123 or in the PCR products of MP124, MP125, MP126 and MP127 previously purifed yesterday by the QIAquick Gel Extraction Kit, we carried out an electrophoresis assay in a 1,5% agarose gel.

• 5 µL of the sample
• 1 µL of 6X blue loading dye

Results : The MP123 plasmid is clearly visible but the PCR products (purified by QIAquick Gel Extraction) aren't. There might be a problem during the purification step using the QIAquick Gel Extraction. Whatever, we still go on with the digestion.

Besides the MP123 sample (J61002 plasmid) was quantified using a Bio Photometer (Eppendorf).

Digestion

For each sample (MP123 and PCR products of MP124, MP125, MP126 and MP127):

• 10 µL of DNA (Miniprep eluate or PCR products)
• 12,5 µL water
• 2,5 µL buffer 2
• 0,25 µL BSA
• 1 µL EcoRI

Incubation 1h30 at 37°C
Incubation 1h30 more after adding 1 µL of SpeI
Electrophoresis of the digested J61002 plasmid
Excision, dissolution and purification of the band of interest using the Wizard SV Gel and PCR Clean-Up System (Promega).
Purification of the digested PCR products using the same kit.

Analysis of the purified digested DNA:

Ligation