IGEM:Harvard/2009/Notebook/Harvard iGEM 2010/2010/07/26
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PFX polymerase PCR
9.5μL + 4μL = 13.5μL therefore 6.5μL H20 per reaction
Making LB+Kan Plates
15μL Kanamycin 30μL DH2O to ease streaking
Streaked this on LB agar plates with glass beads, and then turned the plate upside down and let dry until it was ready for bacterial sequencing, and then those same beads were reused.
Transformation of pLAS and Terminator
Resuspended the following from the registry with 10μL DH2O
p(LAS)TetO, on pSB2K3 (kan res.), from plate 2, sell 11N Terminator, on pSB1AK3 (kan and amp res.), from plate 1, well 4H
Last week, we finished ligation together Sense and Intron parts of the hpRNA and transformed them into E. coli. This week, we will extract the plasmids, add in the antisense part, and transform.
We also need to check which parts of the sequence on the team page corresponds to the sense/antisense parts of the gene. There may have been mistakes in the past.
Culture We cultured 38 colonies. All grew but one LTP+PAL and Ger+Pal grew. We labeled the cultures 1- 38 for simplicity sake.
1-6: LTP + PDk 7-16: Miniprep
Here are the concentrations of the miniprepped plasmid.
We digested the vectors with Spe and Pst1 and ligate with antisense parts (already digested and gel purified). We ran them on the E Gel too long so the lanes do not show anything conclusive, other than that there is digested DNA in the bottom half.Result:
Sequence review clarified which parts of the sequences on the team page corresponded to sense/antisense.
Digestion Reactions Mira N: 888ng Mira C: 888ng Brazz N: 993ng Brazz C: 1032ng STOP: 781ng x 2 (max DNA available)