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). The number of this plasmid extraction is MP123. This extraction has been conducted 3 times.

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. The ladder used was the 100 bp DNA ladder (New England Biolabs).

• 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

The reaction was incubated 1h30 at 37°C, then 1h30 more after adding 1 µL of SpeI. The digested J61002 plasmid was run in an agarose gel and the band of interest was excised, dissolved and purified using the Wizard SV Gel and PCR Clean-Up System (Promega). The digestion products of the PCR products were purified using the same kit.