Team:Paris/August 6

Plasmid extraction

The J61002 plasmid was extrated from overnight bacteria culture. 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 by QIAquick Gel Extraction yesterday, we carried out an electrophoresis assay in a 1,5% agarose gel. The ladder used was the 100 bp DNA ladder (New England Biolabs). We added 1 µL of 6X blue loading dye on 5 µL of each sample, which were then loaded on the gel.

Results : The MP123 plasmid is clearly visible but the PCR products (purified by QIAquick Gel Extraction)

Moreover the MP123 sample was quantified using a Bio Photometer (Eppendorf).

Digestion

For each sample (MP123 and PCR products of MP124, MP125, MP126 and MP127 previously purified by QIAquick Gel Extraction):

• 10 µL of DNA (Miniprep eluate or Gel extraction eluate of the 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.