Team:Groningen/Labwork/16 July 2013

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Revision as of 14:43, 16 July 2013

Mirjam
Finally the colonies are growing on LB agar plates with ampicillin. The negative control does not show any colonies, the double negative does??, the positive control shows a lot of red colonies. On one of the four plates there are no colonies present, on two of them only a couple and on one a lot.

The colonies are picked and placed in LB medium with ampicillin to grow further for miniprep. Colony PCR revealed no bands.

Mini prep analysis is done with the colonies of plate 1 and 3.
Restriction analysis is done with EcoRV.

Inne
Did a PCR with 3 samples in duplo

Sample	Primer F	Primer R	Annealing temp.
1	without strep tag	without strep tag	78 C
2	with strep tag	without strep tag	78 C
3	without strep tag	with strep tag	80 C

Protocol:

amount	compound
28.2 uL	MilliQ
1.5 uL	5% DMSO
1 uL	each dNTP
10 uL	Phusion GC buffer
2.5 uL	Primer F
2.5 uL	Primer R
1 uL	Silk template BBa_16
0.3 uL	Phusion polymerase

Ran the gel 1.5% agarose using the big slots, this posed a couple of problems:
The gel was to thick, the slots were still open but the electrophoresis machine needed to be filled up to the brim with 1x KBE buffer.
epje 3. (duplo) opened during the PCR so almost no solution was left.
added 60 uL gene ruler to the first slot, which is way to much.
gel ran ADD 90 V for 45 minutes, started foaming because of to much KBE.

made new KBE buffer. 100ml KBE/900ml demiwater.

Sander
did a Restriction digestion for Silk and Signal Sequences. incubation started at 11:00 for the signal sequences and 11:30 for the silk.
at 15:38 the restriction digestion reaction was run over a 1,5 % agarose gel with 100 v for 22 min.

@@ Line 5: / Line 5: @@
 Colony PCR revealed no bands.
-Mini prep analysis is done on the colonies of plate 1 and 3.
+Mini prep analysis is done with the colonies of plate 1 and 3.
+<br/>Restriction analysis is done with EcoRV.
 '''Inne'''