Lab 208

Main purpose

• fix problem with gels
• run gels

Who was in the lab

Kristian, Henrike

Procedure

Running and loading gels.

Results

Gels

• 1 kb
• colony PCR NirG
• constitutive reference promoter, GC buffer
• constitutive reference promoter, GC buffer + 3% DMSO
• constitutive reference promoter, HF buffer
• constitutive reference promoter, HF buffer + 3% DMSO
• 1 kb

Gel could not be read and will be redone.

• 1 kb
• colony PCR cycAX, colony 2
• colony PCR cycAX, colony 3
• colony PCR cycAX, colony 4
• colony PCR cycAX, colony 6
• colony PCR cycAX, colony 7
• colony PCR cycAX, colony 8
• colony PCR cycAX, colony 9
• constitutive reference promoter, GC buffer
• constitutive reference promoter, GC buffer + 3% DMSO
• constitutive reference promoter, HF buffer
• constitutive reference promoter, HF buffer + 3% DMSO
• 1 kb

The insert is confirmed for the colonies 3, 4 and 8.

Gel optimization test

Made two gels with TAE buffer to run on the two different power supplies and used two different loading dyes to try and resolve the problem with the bad gels we've been having.

Loading dyes: Agarose Blue was made by us, GelPilot is supplied by Qiagen. The const ref samples were mixed with agarose blue before, so we cannot test the other loading dye on those.

Run on the old power supply (the grey one):

• 1 kb
• cycAX col 3, agarose blue
• cycAX col 4, GelPilot
• cycAX col 8, GelPilot
• cycAX col 2, agarose blue
• cycAX col 6, agarose blue
• cycAX col 7, GelPilot
• cycAX col 9, GelPilot
• constitutive reference promoter, GC buffer
• constitutive reference promoter, GC buffer + 3% DMSO
• constitutive reference promoter, HF buffer
• constitutive reference promoter, HF buffer + 3% DMSO
• NirG col, agarose blue
• NirG col, GelPilot
• HAO col, agarose blue
• HAO col, GelPilot
• 1 kb

Run on the new power supply (the green one):

• 1 kb
• cycAX col 3, agarose blue
• cycAX col 4, GelPilot
• cycAX col 8, GelPilot
• cycAX col 2, agarose blue
• cycAX col 7, GelPilot
• constitutive reference promoter, GC buffer
• constitutive reference promoter, GC buffer + 3% DMSO
• constitutive reference promoter, HF buffer
• cycAX col 3, GelPilot
• NirG col, agarose blue
• NirG col, GelPilot
• HAO col, agarose blue
• HAO col, GelPilot
• 1 kb

Conclusion

There is no visible difference between the two power-supplies and loading dyes. Probably the TBE we made from powder was not good, we will use TAE from now on.

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