Team:Paris/July 28

DNA Extraction

Results of the extraction : Electrophoresis

conditions of electrophoresis:

• 10µl of ladder 1 kb
• xµl of extraction added with 2µl of loading Dye 6x
• migration ~30min at 100W

• Gel 1, 2, 3, 4 = 0,8%
• 2µl of extraction added with 2µl of loading Dye 6x

• Gel 5, 6 = 0,8%
• 3µl of extraction added with 2µl of loading Dye 6x

• Gel 7 = 0,8%
• 5µl of extraction added with 2µl of loading Dye 6x

• Gel 8 = 1,5%
• 10µl of extraction added with 2µl of loading Dye 6x

Results :

gel1 gel2 gel3 gel4 gel5

gel6 gel7 gel8

==> Conclusion : for most of the samples we have enough signal to determine the concentration of the digestion to do the ligation.

For the samples we don't succeed to obtain signals, we have migrated a second gel ( gel n°7).

==> Conclusion : The gel n°7 allowed us to know the concentration of other digestion undetermined.

Ligation

Protocol

For each samples,

• 1 µl Ligase
• X µl Vector
• Y µl Insert (3x more than vector)
• 2 µl Ligase Buffer 10x
• 20 µl qsp H2O

• Incubates O/N at 4°C (in the fridge)

PCR

Protocol

For each samples (MP 100 and MP 120),

• 1 µl dNTP
• 10 µl Buffer Phusion 5x
• 2,5 µl VR2 (O18)
• 2,5 µl VF (O19)
• 1 µl Phusion
• 50 µl qsp H2O (33µl)

When the PCR cycles were finished, 10 µL of 6X loading dye were added. The samples were then loaded (2 x 30 µL per sample) on a 1,5% agarose gel. After electrophoresis, the bands corresponding to MP 100 and MP 120 were excised and purified using the QIAquick DNA Gel Extraction Kit (QIAGEN). The elution was made in 50 µL of water. Because the intensity of the band corresponding to MP 120 was very low, we only continued with MP 100. MP 100 was digested by EcoRI & SpeI (Forward Insert) or by XbaI & PstI (D100 : Backward Insert).

Digestion reaction (total volume : 50 µL)

• 25 µL DNA
• 5 µL buffer 2 (10X)
• 2 µL enzyme 1
• 2 µL enzyme 2
• 0.5 µL BSA (100X)
• 15,5 µL water

The reaction was incubated 2 hours at 37°C. The samples (2 x 30 µL per sample) were then analysed by electrophoresis.