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Revision as of 18:22, 19 July 2013 (view source) Gosia (Talk | contribs) (→Procedure) ← Older edit		Revision as of 18:23, 19 July 2013 (view source) Gosia (Talk | contribs) (→Restriction analysis) Newer edit →
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	==Results==		==Results==

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Revision as of 18:23, 19 July 2013

Contents 1 208 1.1 Main purpose 1.2 Who was in the lab 1.3 Procedure 1.3.1 Plasmid isolation 1.3.2 Colony PCR 1.3.3 Restriction analysis 1.4 Results 1.5 Conclusion

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Main purpose

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• plasmid isolation
• colony PCR to verify transformants from 18-07-2013.
• gel electrophoresis to verify insert after colony PCR (AMO, HAO), plasmid isolation
• gel electrophoresis to verify plasmid isolation
• restriction analysis of isolated plasmids
• gel electrophoresis to verify restriction analysis

Who was in the lab

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Gosia, Henrike

Procedure

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Plasmid isolation

According to protocol in QIAprep Spin Miniprep Kit

Colony PCR

Master mix was prepared based on standard colony PCR procedure.

For AMO primers 29a, 29b were used (no USER primers this time) and for HAO primers 28a, 28b were used. We used Phusion polymerase. We obtained 7 transformants after yesterday transformation with AMO in pZA21 and 3 transformants which may contain HAO in pZA21. On negative control plate 3 colonies grew.

PCR parameters according to standard reaction with differences in annealing temperatures (for AMO -> 54°C, for HAO -> 56°C) and with 3 min of extension time. Expected fragments lengths are AMO - 3074 bp, HAO - 2816 bp.

Restriction analysis

Restriction analysis was performed with EcoRI enzyme.

Compound	Volume uL
DNA (miniprep)	10
Buffer for EcoRI 10x	2
water	7,5
EcoRI enzyme	0,5

Incubation for 1-2 hours at 37°C.

Results

1% gel of results of today's colony PCR and yesterday's PCR to add his-tags

• 1: HAO, colony PCR
• 2: HAO, colony PCR
• 3: HAO, colony PCR
• 4: AMO, colony PCR
• 5: AMO, colony PCR
• 6: AMO, colony PCR
• 7: AMO, colony PCR
• 8: AMO, colony PCR
• 9: 1kb ladder
• 10: AMO, colony PCR
• 11: AMO, colony PCR
• 12: TAT construct, his-tag, program ramp1
• 12: TAT construct, his-tag, program ramp2
• 12: Sec construct, his-tag, program ramp1
• 12: Sec construct, his-tag, program ramp2

1% gel of plasmids isolated by qiagen spin miniprep kit

• 1: 1kb ladder
• 2: cycAX from isolation of the 19-07
• 3: cycAX from isolation of the 19-07 dub
• 4: cycAX from isolation of the 17-07
• 5: RFP from isolation on the 19-07
• 6: RFP from isolation on the 19-07 dub
• 7: RFP from isolation on the 19-07 trip

Conclusion