IGEM:Harvard/2006/Cyanobacteria/Notebook/2006-8-1: Difference between revisions
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**#* This ligation can produce backwards insertions and tandem insertions (e.g. KaiA+KaiA), but we can screen them out later when we do our X and P digest. | **#* This ligation can produce backwards insertions and tandem insertions (e.g. KaiA+KaiA), but we can screen them out later when we do our X and P digest. | ||
**#* At this point we can transform and amplify our KaiA/B inserts. | **#* At this point we can transform and amplify our KaiA/B inserts. | ||
**# | **# After we've amplified the plasmid, digest the resulting plasmid at X and P (or E and S). | ||
**# Run the result on a gel to screen for our construct. | **# Run the result on a gel to screen for our construct. | ||
Revision as of 12:56, 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.
- Biobrick assembly of KaiA and KaiB
- Preparation of backbone
- Nick has prepared high-copy plasmids for us to use as backbones (they currently have an RFP insert which must be removed).
- Miniprep the cultures that Nick provided
- Digest the plasmids at E and P.
- Run the digested plasmids on a gel to separate the backbone from the insert (and also run some undigested plasmid for comparison).
- Gel-extract the backbone.
- Add phosphatase to the backbone to prevent self-ligation (NOTE: this step might have to be done earlier).
- Ligation of KaiA/B with backbone
- Gel purify the KaiA and KaiB bands
- Digest KaiA/B at X and S.
- Ligate KaiA/B inserts with the backbone prepared above.
- This ligation can produce backwards insertions and tandem insertions (e.g. KaiA+KaiA), but we can screen them out later when we do our X and P digest.
- At this point we can transform and amplify our KaiA/B inserts.
- After we've amplified the plasmid, digest the resulting plasmid at X and P (or E and S).
- Run the result on a gel to screen for our construct.
- Preparation of backbone
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
