IGEM:Harvard/2006/Cyanobacteria/Notebook/2006-8-1: Difference between revisions
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NOTE: Raw PCR products of interest were put in .5mL tubes in the freezer in a box labeled with 8.1. | NOTE: Raw PCR products of interest were put in .5mL tubes in the freezer in a box labeled with 8.1. | ||
==Gel images from yesterday's split PCR== | ==Gel images from yesterday's split-extraction PCR== | ||
[[Image:2006-8-1 split PCR.jpg|thumb|left|Click for legend]] | [[Image:2006-8-1 split PCR.jpg|thumb|left|Click for legend]] | ||
It looks like our KaiA and KaiB extractions succeeded, but KaiC did not. | |||
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Revision as of 12:41, 1 August 2006
Gel images from yesterday's mutagenesis PCR #7
It looks like the 3-10 and 5-4 mutagenesis PCRs succeeded, but the 7-6 and 9-8 did not. Also, we got a strange 400b band from the crossF + crossR reaction instead of the 3kb we predicted. The band is reminescent of the 400b band we saw from genomic extraction #5
NOTE: Raw PCR products of interest were put in .5mL tubes in the freezer in a box labeled with 8.1.
Gel images from yesterday's split-extraction PCR
It looks like our KaiA and KaiB extractions succeeded, but KaiC did not.
Plan for the next few days
We're going to do BioBrick assembly of the KaiA and KaiB we extracted last night, keep trying to extract KaiC from the cyanobacteria genome, investigate the goofy 400b band, and try to get the other mutagenesis reactions to work.
Extracting longer segments via gradient pcr (mutagenesis #8)
As during the last PCR we were able to get 2 of the 4 segments and we got a 400bp band for "all" which was similiar to the colony PCR band, I hypothesize that a gradient PCR may show us a 3kb "all" band as well as our other products. Hotstartaq as usual.
Vials
- 9-8, 6-7, all, -t for each, cat gene as a positive control, master mix
- 6 for each + negative
- 24 total vials
For cat gene, pbc_ks+, kt_b, kt_bb primers - expected size ~1.1kb
Back to basics: HotStarTaq/Farren Protocol
5 µL 10x buffer (HotStar) 1.0 µL 10 mM dNTPs 1.0 µL 20 µM forward primer 1.0 µL 20 µM reverse primer 1.0 µL plasmid DNA 0.5 µL HotStarTaq DNA polymerase 40.5 uL dH20
PCR Time
#*95 °C for 15 min. (melt) #*95 °C for 0.5 min. (melt) #*GRADIENT °C for 0.5 min. (anneal) #*72 °C for 2.5 min. (extension) #* Go to step 2 (30x) #*72 °C for 10 min. (final extension) #* Keep at 4 °C forever
Total time: 111 minutes +/- some = 120min
