Team:Stockholm/23 July 2010

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Revision as of 22:24, 25 August 2010

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.

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.

@@ Line 1: / Line 1: @@
 {{Stockholm/Top2}}
+==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:
+[[Image:Bild23.jpg]]
+===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:
+[[Image:Bild24.jpg]]
+[[Image:Bild25.jpg|250px]]
+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:
+[[Image:Bild26.jpg]]
+[[Image:Bild27.jpg|250px]]
+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.
+----
 ==Johan==