IGEM:Harvard/2009/Notebook/Harvard iGEM 2010/2010/07/27
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Yesterday, we tried to run a diagnostic digest of the Sense + Intron hpRNA. The gel did not work properly, so we are redoing the gel today. We'll also go ahead and ligate antisense parts into all 38 minipreps that we HOPE contains correct Sense + Intron. Once the gel finishes running, we can go back and dispose of the ligations today that do not contain the correct S+I parts.
Today, we are also going to redo the ligation of Sense + Intron for PDK colonies because the gel that we are redoing today suggests that no PDK worked from last week's ligation.
Redoing ligations for Sense + PDK
38 digests of the sense+intron miniprepped plasmid DNA. The digest was done with Slow Digest Eco and Pst
From the digest, we can see that two LTPS+Pal worked (9,11), 3 Ger3S+Pal worked (36-38), 4 Bet2S+Pal Worked (25-28)
Lanes are: 1-6: LTP + PDK (3200 & 1200);
7-16: LTP + PAL (3200 & 540);
17,18: Bet1 + PDK (3200 & 1200);
19-22: Bet1 + PAL (3200 & 540);
23,24: Bet2+PDK (3200 & 1200);
25-28: Bet2 + PAL (3200 & 540);
29-32: Ger3 + PDK (3200 & 1200);
33-38: Ger3 + PAL (3200 & 540);
Image: Ladder (every 6)
Rerun of the lanes that were ligations of PDK + sense because they did not show on the gel above. We feel that there might be a chance that if the Sense+PDK assembly did not work, the 300bp evidence ran off the first gel. This second gel shows that most of the PDK+sense did NOT work. Only three worked -- minipreps 16, 30, and 31.
Digested antisense parts with X, P and pSense+Intron with S, P. Digestion ran for 2 hours, with an additional 20 minutes to de-phosphatize the backbone. Ligations were done for colonies that showed correct Sense+Intron ligations.
Transform and Plate
The standard Turbo Cell transformation protocol was followed.
Redo Ligations for PDK+ Sense
Since the gel showed that most of them did not work.
Digestion of pLTP, pBet v1, and pGer 3 with S, P to prepare them as a back bone. Phosphatase was used. Did not digest pBet v2 because there was not enough.
PCR purify the digested backbones containing Sense.
pLTP - 24.0 pBet - 17.2 pGer - 42.8
Ligate predigested PDK digested with X, P and ligate into purified backbone. Transform and Plate (6? plates total)
Ligation of Mira/Brazz + StrepII to STOP + V0120
Miraculin: 34ng Brazzein: 11ng Backbone: 50ng
PCR of Valencene using Pfx Polymerase
Specs: DNA 1-1: 1.8 ng/μL DNA 1-2: 1.1 ng/μL DNA 2-1: 2.6 ng/μL DNA 2-2: 2.2 ng/μL
Mira/Brazz+StrepII+STOP confirmation digest
Digested DNA: MN2: 211.2 ng MC2: 190.2 ng BN2: 184.4 ng BC2: 219.2 ng
(these are all amp resistant)
The same is done in LB+Kan for pLAS and Act2lacO promoter, which are Kanamycin resistant.
Redoing Barnase-strep Tag Ligation
poured a 2% agarose gel to gel-purify the barnase from Barnase1 Xba Pst1 cleanup and Barnase2 Xba Pst1 cleanup, both from (7/23)
Phosphatase Treating B15 Spe1/Pst1 Cleanup