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Team:University of Lethbridge/Notebook/GeneralLabAugust
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August 5, 2008
Nathan Puhl et al
Gel extracted two bands really closer to each other from PCR (August 1) at ~76 bp. Used Qiagen MiniElute kit. Results are in the hard copy lab notebook. Seems as if gel extraction was successful. ____ 2 color reporter system:
Set up digest of LacI and DT.
LacI - EcoRI and SpeI
DT - EcoRI and XbaI
Reaction mixture:
-5 uL template -5 uL NEB 2 Buffer -0.5 uL BSA? -1 uL RE #1 -1 uL RE #2 - 37.5 uL ddH20
Left at 37 C overnight. Heat deactivation (65 C) for 15 minutes.
August 6, 2008
Nathan Puhl, Roxanne
2 color reporter system:
Gel extracted LacI insert and DT vector. Didn't run gel yet.
August 13, 2008
Nathan Puhl and Sebastian
Verified and quantified the LacI and DT restriction digest products.
Set up a Ligase experiment for LacI and DT
- 1.3 uL vector(DT) - 8 uL insert(LacI) - 2 uL 10x Ligase buffer - 1.5 uL Ligase - 7.2 uL ddH2O - 20 uL total volume
August 14, 15
Nathan Puhl, Munima, Selina
Poured 61 LB + Amp plates. Stored in iGEM 4 C fridge.
August 15, 2008
Roxanne
Used Qiagen Plasmid MiniPrep Kit to Plasmid Prep Last Year's Biobrick Parts that were Incubated overnight.
Ran Plasmids in a 1% Agarose Gel at 100 V for 30 minutes.
Christa, Munima, Sebastian
Did a plasmid prep on pSB1A7 using QIAprep Spin Miniprep Kit. Stocked 4- 50uL of pSB1A7 and stored in iGEM -20 C.
Sebastian, Nathan Puhl
Ran products from plasmid prep (pSB1A7 x 4 samples) on 1% agarose gel for 27 minutes.
-Lane 1 - 1 kb GeneRuler ladder (2 uL) -Lane 2 -6 pSB1A7 (3 uL) + 6x loading dye (2 uL); Mixed up what sample was in Lane 4, so Lanes 5 and 6 were run.
Conclusion: The bands appeared to be at the correct size for pSB1A7.
August 21
Nathan Puhl, Roxanne
-Screened the pSB1A7 + RS1, and pSB1A7 + RS2 by PCR using the VF2 and RS1/RS2 Reverse Primers determine whether the plasmids obtained from the recombinant cells contain the riboswitch, and if so, if it inserted in the correct orientation.
August 21, 2008
Roxanne
-Ran the PCR products on a 1% Agarose Gel at 100 V for 33 minutes. The gel was empty with the exception of primer dimers.
Nathan Puhl, Roxanne
-went over the SELEX protocol with HJ to determine the primers we will need to do this, and how exactly we plan on perfoming the evolution.
-setup a restriction digest for pSB1A7 using XbaI and SpeI, ran overnight.
August 22, 2008
Christa, Munima, Nathan Puhl, Roxanne
Objective: Run a gel to confirm that appropriate inserts were amplified from the PCRs and do a gel extraction of the inserts to prepare them for the biobrick format.
-Could not obtain a picture of the gel (1% agarose) of the half of the digested pSB1A7 (15 uL x 3 wells) and the recently amplified CheZ gene (15 uL x 3 wells) because the camera would not turn on. The CheZ gene appeared at the correct size (~700 bp). -Did a gel extraction of pSB1A7 and CheZ from that gel with the Qiagen MiniElute Gel Extraction kit. Final volume of each was 10 uL. -Ran another 1% agarose gel to confirm that the gel extraction was successful. Ran 1 uL of each sample. Bands appeared at appropriate sizes. The concentrations of pSB1A7 and CheZ were estimated to be 25 ng/uL and 80 ng/uL, respectively.
Next step: Digest pSB1A7 with antarctic phosphatase. A restriction digest will be performed at a later date on CheZ to prepare it for insertion.
August 23, 2008
Nathan Puhl, Roxanne
-Digested pSB1A7 with Antarctic Phosphatase
-9 uL of cut pSB1A7 -1.5 uL of 10x Antarctic Phosphatase Buffer -1 uL of Antarctic Phosphatase Enzyme -3.5 uL of water Allowed the Reaction to take place for 30 minutes to remove the 5` Phosphates from the pSB1A7 plasmid to prevent religation.
-Ran the remainder of the pSB1A7 plasmid from August 22nd on 1 1% Agarose Gel at 100 v for 27 minutes.
-Gel Extracted the plasmid DNA.
-Purified the Phosphatase reaction to isolate the pSB1A7 DNA.
-Ran a 1% gel to quantify the amount of plasmid DNA present.
-Ligated RS1 and RS2 into the dephosphorylated pSB1A7 using T4 DNA Ligase.
-1 uL of RS1 or RS2 -4 uL of dephosphorylated pSB1A7 -1 uL of 10X T4 DNA Ligase Buffer -0.33 uL T4 DNA Ligase Enzyme -3.67 uL water Reaction was allowed to go overnight
August 24, 2008
Nathan Puhl
-Transformed DH5a cells with the RS1+pSB1A7 or RS2+pSB1A7 plasmid on semi-solid agar plates containing 100 ug/mLof ampicillin.
August 26, 2008
Roxanne, John
-Restriction Digested the Biobrick Parts I13504, I13401, P0440, C0014, B0015, J31007 with XbaI and PstI
-20 uL template (~2 ug) -5 uL React 2 -4 uL XbaI -4 uL PstI -17 uL ddH2O ____ 50uL Reaction in 37.0C H2O bath overnight
August 27, 2008
Roxanne
-Inactivated the Restriction Enzymes by placing them on the heating block at 65.0C for 10 minutes -Ran 25uL of DNA on a 1% Agarose Gel at 100 V for 30 minutes -Gel Extracted the DNA -Ran 1 uL of DNA in a 1% Agarose Gel at 100V for 30 minutes.
August 28, 2008
Roxanne, Munima, Sebastian, Nathan Puhl
-Performed a restriction digest on LacI+dt, pLacI, pStrong, and RFP sub
-10 uL template -5 uL NEB 2 -2 uL Restriction Enzyme #1 (Xba I or Spe I) -2 uL Restriction Enzyme #2 (Pst I) -21 uL ddH20 ____ 50 uL Rxn left to run overnight at 37.0
-Ran a Gel of the Riboswitch PCR which had been done with Taq a couple of weeks ago to quantify the amount of DNA present. Setup the Math to perform a ligation into pGEM T-easy.
-Picked a colony from the dT plate which was stored in the fridge from several weeks ago, incubated overnight at 37.0C
August 29, 2008
Roxanne, Nathan Puhl, Munima, Sebastian, Andrew
-Inactivated the Restriction Enzymes, and performed a gel extraction of the parts restricted on Aug. 28.
-Plasmid prepped and glycerol stocked the dT part which had been incubated the night before.
-Performed the ligation of the riboswitch into pGEM T-easy, transformed and plated.
=Roxanne
-Gel Extracted the remainder of the DNA
August 30, 2008
Nathan Puhl, Roxanne, Andrew
-Performed a colony PCR on representative colonies containing the pGEM T-easy plasmid to screen for the presence of the riboswitch.
-Ran a gel of the PCR products on 3% Agarose @ 100V for 27 minutes.
-Ran a gel of the Digested Parts on 1% Agarose @ 100V for 27 minutes.
