IGEM:Harvard/2009/Notebook/Harvard iGEM 2010/2010/07/27

{| width="800"
 * style="background-color: #EEE"|[[Image:igem-logo-150px.png|150px]] iGEM iGarden
 * style="background-color: #F2F2F2" align="center"|  |Main project page
 * style="background-color: #F2F2F2" align="center"|  |Main project page


 * colspan="2"|
 * colspan="2"|

Procedures
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.

Procedures
 * 1) Diagnostic Digest
 * 2) Diagnostic Gel
 * 3) Diagnostic Gel of PDK + Sense
 * 4) Ligation of Antisense Part into 38 minipreps

Redoing ligations for Sense + PDK
 * 1) Digest of pLTP, pBet v1, pGer with S, P
 * 2) PCR purification
 * 3) Ligation of Sense+PDK
 * 4) Transform and Plate

Results
Diagnostic Digest

38 digests of the sense+intron miniprepped plasmid DNA. The digest was done with Slow Digest Eco and Pst

Diagnostic Gel

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.



Ligated with antisense parts and transformed

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.

Nanodrop

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
 * Insert to Backbone ration of 3:1


 * Ligations were transformed, plated onto LB+AMP and left overnight @ 37°C

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
 * Pfx polymerase was rumoured to be more accurate at PCR from genomic DNA


 * 1μL of each DNA sample was used for PCR. Cycling procedure can be found here: http://www.invitrogen.com/etc/medialib/en/filelibrary/pdf.Par.79411.File.dat/platinumpfx_pps.pdf

Mira/Brazz+StrepII+STOP confirmation digest
Digested DNA: MN2: 211.2 ng    MC2: 190.2 ng     BN2:  184.4 ng     BC2:  219.2 ng


 * Digestions were run on a 1% agarose gel at 125V for 30 minutes.


 * 1) Ladder
 * MN2
 * MC2
 * BN2
 * BC2
 * 1) Ladder

Innoculations
(these are all amp resistant)
 * RXRHuman, RXRLocust, Barn1strep, Barn2strep, ACC, and EcR
 * 6 colonies of each are inocculated, except for Barn1 and Barn2, of which 4 are innocculated each.
 * 5mL LB+amp and a smear of each colony from a relevant plate

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)


 * Gel ran empty except for DB ladders

Phosphatase Treating B15 Spe1/Pst1 Cleanup

 * to prevent religation of the whole vector


 * 35μL B15 Spe1 Pst1 cleanup from 7/23
 * 4μL 10x green FD buffer
 * 1μL Alkaline phosphatase


 * }