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From 2010.igem.org

Contents 1 Nina 1.1 Mini prep on IgG protease 1.2 Colony PCR on CPP 2 Johan 2.1 Colony PCR 2.2 Gel electrophoresis

Nina

Mini prep on IgG protease

I did a mini prep on the IgG protease inserted in the bank vectors A,C and K. The procedure was according to the method described in protocols.

• vector A: colony #5
• vector C: colony #10
• vector K: colony #2

Spectrophotometer:

Colony PCR on CPP

I performed a colony screen on the two dishes called + (with insert) and - (without insert) 8. I picked five colonies from each dish.

PCR Master mix 10 tubes:

• Mgcl2 50 mM 10 ul
• Phusion buffer 5X 100 ul
• dNTP 10 mM 10 ul
• primer VF2 10 uM 30 ul
• primer VR 10 uM 30 ul
• PjuX7 10 ul
• H2O 300 ul

I added 49 ul to each PCR tube. Before adding the mixture to each PCR tube I transfered about half of a colony of interest into the tube and microwaved it for 1 min. This is to lyse the bacteria before PCR it.

Ladder: FastRuler™ Ultra Low Range DNA Ladder, ready-to-use, 10-200 bp Fermentas

Arragement on gel:

Colony nr 9 and 40 seem interesting to send for sequencing since they are bigger in size than the other bands.

Ladder: FastRuler™ Ultra Low Range DNA Ladder, ready-to-use, 10-200 bp Fermentas

Arragement on gel:

This gel should have shown all the bands in the same size, however they do not , but still they are not as big as the bands on the plus 8 gel in the colonies # 9 and 40. This might indicate that there is something interesting with these colonies, which thus will be sent for sequencing.

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Johan

Colony PCR

• Another colony PCR on the second transformation of TAT CCP as the first one didn't show many colonies and none of the colonies showed any insert in the vector.

• Colony #1-36 +insert, colony #42-43 -insert.

• PCR reaction mix
  • 0,5 µl Pfu polymerase
  • 0,5 µl 10 mM dNTPs
  • 2 µl 10 µm f.primer (VF2)
  • 2 µl 10 µm r.primer (VR)
  • 2,5 µl Pfu buffer 10x
  • 17,5 µl H2O

• PCR program
  • 98 °C - 2 min
  • 35 cycles of
    • 98 °C - 10 sec
    • 55 °C - 15 sec
    • 72 °C - 45 sec
  • 72 °C - 5 min
  • 4°C - ∞

Gel electrophoresis

A gel electrophoresis was performed on the PCR products.

Lane 1 & 40: FastRuler DNA Ladder low range, lane 2-37: +insert, lane 38-39: -insert.

The gel didn't turn out good, probably I used too much DNA template.