Team:Stockholm/18 July 2010

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(New page: {{Stockholm/Top2}} ==Andreas== ===BL21 restreaks=== right ''Results from 17/7 restreaks'' Nice fluorescent cell colonies a...)
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Nice fluorescent cell colonies appeared on the plate, indicating that IPTG induction from the pEX vector indeed works and that I have successfully inserted the three fluorescent protein genes into pEX.
Nice fluorescent cell colonies appeared on the plate, indicating that IPTG induction from the pEX vector indeed works and that I have successfully inserted the three fluorescent protein genes into pEX.
 +
 +
===Competent Top10===
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''Continued from 17/7''
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At 9:30 AM cell culture still hadn't reached OD<sub>600</sub> 0.3, only 0.02. I therefore transfered the cells to 37°C, 250 rpm until an OD<sub>600</sub> of 0.3. I then followed our standard Top10 protocol.
 +
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===Colony PCR of pEX constructs===
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''Continued from 17/7''
 +
 +
A new colony PCR was run on the same clones as were picked 17/7, using the same tube settings except (1) 1.5 &mu;l primer was used instead of 1.0 &mu;l and (2) 1 &mu;l cell suspension was used instead of 0.5 &mu;l; dH<sub>2</sub>O volume was decreased accordingly. Samples pEX.SOD A and pEX.SOD B were ommitted.
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'''PCR settings'''
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# 95&deg;C - 5:00
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# 95&deg;C - 1:00
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# 60&deg;C - 0:30
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# 72&deg;C - 1:15 (Cycle to step 2. 29 times)
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# 72&deg;C - 5:00
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# 15&deg;C - &infin;
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====Gel verification====
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1 % agarose, 90 V, 40 min
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''Results''
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Not all bands present in all lanes. Due to lack of time, all samples were discarded and I will pick/verify new colonies after my vacation.

Revision as of 20:56, 29 July 2010

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Contents

Andreas

BL21 restreaks

IPTG-induced BL21

Results from 17/7 restreaks

Nice fluorescent cell colonies appeared on the plate, indicating that IPTG induction from the pEX vector indeed works and that I have successfully inserted the three fluorescent protein genes into pEX.

Competent Top10

Continued from 17/7 At 9:30 AM cell culture still hadn't reached OD600 0.3, only 0.02. I therefore transfered the cells to 37°C, 250 rpm until an OD600 of 0.3. I then followed our standard Top10 protocol.

Colony PCR of pEX constructs

Continued from 17/7

A new colony PCR was run on the same clones as were picked 17/7, using the same tube settings except (1) 1.5 μl primer was used instead of 1.0 μl and (2) 1 μl cell suspension was used instead of 0.5 μl; dH2O volume was decreased accordingly. Samples pEX.SOD A and pEX.SOD B were ommitted.

PCR settings

  1. 95°C - 5:00
  2. 95°C - 1:00
  3. 60°C - 0:30
  4. 72°C - 1:15 (Cycle to step 2. 29 times)
  5. 72°C - 5:00
  6. 15°C - ∞

Gel verification

1 % agarose, 90 V, 40 min

Results

Not all bands present in all lanes. Due to lack of time, all samples were discarded and I will pick/verify new colonies after my vacation.

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