Restriction, Ligation, and Transformation Lumazine synthase - double terminator and pBAD - RBS into pSB1C3
1. Restriction
2. Ligation
Picked cells from June 21st transformation
Minipreped Cells picked on June 25th, 2011
Restriction digest and ran on gel
Lumazine - dt construct successfully inserted into pSB1C3
Glycerol stocked.
Add Gel Picture
Performed antibiotic assay on lumazine - dt in pSB1C3
Samples from one colony grew in Kanamicyn; this stock was discarded
Ran samples on Gel to confirm sizes
PCR'd out Lumazine Synthase - Dt and RFP
Ran on a 1% agarose gel for 45min at 70volts
Ran 4 samples on an agarose gel:
D22
T22
D24
T24
Preformed a restriction with EcoRI and PstI for Ryan
Ran on 1% agarose gel
Colony PCR of:
k331033-k331035 in pSB1C3
P0440-K331033 in pSB1A2
J04500-LUMAZINE SYNTHASE-DT in pSB1AK3
pBAD-P0440 in pSB1A2
LUMAZINE SYNTASE-DT-pBAD in pSB1A2
Ran PCR samples from July 28th on a 1X TBE Agarose gel
For control comparision of parts from July 28th PCR we ran a PCR of:
K331035
K331033
Lumazine synthase - dt
P0440
Control
EcoRI Restriction of:
J04500-Agn 43 sample 1
J04500 -Agn 43 Sample 2
J04500 - Lumazine synthase - Dt sample 3
K346007
Lumazine synthase - Dt
Ran Restriction on gel to confirm sizes
Notebook team 3 insert picture Page 37
Transfer of enhanced lumazine synthase from pET-28a into pSB1C3
Restricted with EcoR1 and Pst1
Ran on 1% TAE agarose gel for 90 minutes at 80V
colonies picked see lab book team 3/ team 1 pg.42
Gel ran at 80V for 90 minutes
Gel Extraction performed
Transfer of p0440 - K331033 from pSB1A2 into pSB1C3
Ran on 1% TAE gel for 90 minutes at 80V
Transfer of p0440 - K331033 from pSB1A2 into pSB1C3
Ran on 1% TAE gel for 90 minutes at 80V
Ran Restrictions on 1X TAE agarose Gel
Performed a successful Gel extraction
Ligation
performed a miniprep of August 4th transformation, pBAD+p0440 and Lumazine Synthase-Dt + pBAD
Ran a 1% TBE agarose Gel Results of Gel look good
Transformed Enhansed Lumazine Synthase in pSB1C3 and plated
Picked colonies
Religated Enhansed Lumazine synthase into pSB1C3
Miniprep'd overnight cultures from august 8th
Transformed enhansed lumazine synthase into Competent Dh5Alpha Ecoli cells
Restricted J04500 - Lumazine-Dt out of pSB1AK3 and P0440 K331033 out of pSB1A2 With EcoR1 and PstI
Picked Colonies for overnight culture, from August 8th plating of Enhansed Lumazine synthase
Miniprep
Miniprep'd Enhansed Lumazine from over night culture on August 9th.
Made 1% TAE Agarose Gel set in fridge for use next day.
Ligation
Ligated together
Transformation
Transformed ligation mix into DH5Alpha E.coli Cells
Ran Gel from August 10th, fridge keeps gel well.
Picked Colonies from Gylserol stocks
Gel Extraction of J04500 - Lumazine synthase - Dt and P0440 - K331033
Parts Liagated into pSB1C3 and Control Ligation performed with restricted pSB1C3 and ddH20
Ligations were transformed into competent DH5alpha E.coli Cells and Plated
Repeated August 11th, Ligations and Transformations of of J04500 - Lumazine synthase - Dt and P0440 - K331033
Restriction Restricted parts with respective restiction enzymes
Made a 1X TAE Agarose Gel stored in fridge overnight.
Ran Agarose Gel from September 8th.
Gel extraction went well all pieces showed up with the exception of J04500-Enhansed Lumazine in pSB1AK3
Set up ligations of
Assembly of Ptet-Rbs in pSB1A2 to have XylE- ATag-Dt Did a restriction Digest, Cutting pTet-rbs with SpeI and PstI Restricted XylE Atag- dt with XbaI and PstI While the Restricted at 37 degrees Celcus for 1 hour made a 1% TAE Agarose Using TAE from Weiden stocks for both Gel and buffer. Loaded gel with samples and ran at 80Volts for 60 minutes. Was very happy to see the gel running at normal Amps and reaching regular Volts, No more melting Gels!! Gel analysis by UV indicated Restrictions did not work for XylE
3 days back and nothing has worked, Persistence is the only way Were going to get through this!! Build a new copy of Xyle + S04261 Since the stock copy is unaccounted for/Incognito. Found Prerestricted Xyle and S04261 in team ones book, and located in freezer, Performed a ligation, left overnight. Restricted Enhansed Lumazine Synthase-Dt in pSB1C3 cut at EcoRI and XbaI to later ligate with previously restricted J04500 cut at EcoRI and SpeI Also Restricted, J04500 out of pSB1AK3 by cutting at EcoRI and SpeI to be ligated with the Enhansed Lunazine synthase-dt in pSB1C3 Also Restricted Enhansed Lumazine Synthase in pSB1C3 at sites SpeI and PstI and Dt D6 was restricted at XbaI and PstI. ( later to learn should have left Dt in plasmid and removed the enhansed Lumazine synthase, Dt was too small and ran off gel.
Ran all parts on a 1% TAE agarose Gel,
Ligated each of the Enhansed Lumazine Synthase-Dt with The Gel restricted J04500 and the previously restricted J04500
Transformed and plated cells left in incubator overnight
End of the first Week worth of classes, and still nothing grows/ works Why?? I am informed I am using the wrong enzymes, a type that wont heat kill, ( later questioned, but im running a gel for extraction, Does this still matter? Since I was informed the heat kill process doesn’t need to be done when running a gel for extraction, When I was introduced to the gel extractions, I asked why are we doing it this way, the answer was ‘It’s more efficient!’ apparently in theory only) Ok one More Kick at it, New enzymes check, Proper TAE check, advice from Phd students Check, Objective: Get something to work, Feeling I have it all figured now, I set forth once more.
Made Gel while Restrictions incubated,
Stored restricted DNA in Freezer overnight to be used in gel in the morning,
Gel froze overnight in fridge, so Made two new gels 1 small one large both 1% TAE Agarose ( so much for loading a gel and going to class) Added loading dye to samples and ran on gel, Ran gels at 80Volts for 90 minutes Enhansed Lumazine Synthase –dt did not work restriction issue J04500 –Enhansed Lumazine synthase did not work restriction issues XylE –ATag-Dt was not run on gel, portion too small for successful gel extraction/ not enough lanes, Only Enhansed Lumazine and Dt D6 in pSB1A2 were available to proceed to gel extraction, Limited number of Spin columns available, Gel Extraction Failed Wrong product poured into wrong spin column
Pack up go Home Try again in a few days
"
