Team:Groningen/Labwork/16 July 2013

From 2013.igem.org

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<br> Ran the gel 1.5% agarose using the big slots, this posed a couple of problems:
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<br> Ran the gel 1.5% agarose using the big slots,  
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<br> 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.
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<br> gel ran ADD 90 V for 45 minutes.
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<br> epje 3. (duplo) opened during the PCR so almost no solution was left.
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<br>gel didn't run nice, tried to cut out bands from 2 and 3.
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<br> added 60 uL gene ruler to the first slot, which is way to much.
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<br> did gel purification
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<br> gel ran ADD 90 V for 45 minutes, started foaming because of to much KBE.
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<br> Ran 0.8% agarose gel for 24 min on 100 V
<br>made new KBE buffer. 100ml KBE/900ml demiwater.
<br>made new KBE buffer. 100ml KBE/900ml demiwater.

Revision as of 14:50, 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,
gel ran ADD 90 V for 45 minutes.
gel didn't run nice, tried to cut out bands from 2 and 3.
did gel purification
Ran 0.8% agarose gel for 24 min on 100 V


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.

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