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)

Purification of the B0034 and B0030 BioBricks

We performed PCR on the B0034 BioBrick (MP 100) and the B0030 BioBrick (MP 120) to amplify the sequence in order to have enough amount of DNA to carry out the following of our experiments.

PCR Protocol

For each samples,

• 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)

Electrophoresis - Purification

When the PCR cycles were finished,

• Add 10 µL of 6X loading dye.
• Load the samples (2 x 30 µL per sample) on a 1,5% agarose gel.
• Migration 30' at 100W.

After electrophoresis,

• Excision and purification of the bands corresponding to MP 100 and MP 120, using the QIAquick DNA Gel Extraction Kit (QIAGEN).
• Elution in 50 µL of water.

Because the intensity of the band corresponding to MP 120 was very low, we only continued with MP 100.

DNA Digestion

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.

Electrophoresis

The sequence of MP 100 (B0034) digested by EcoRI & SpeI (35 bp) or XbaI & PstI (34 bp) was too short and we didn't manage to visualise it on the gel.

==> Conclusion : small parts like B0034 can't be cloned as an insert.