Team:University of Lethbridge/Notebook/GeneralLabAugust

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(Aug. 1- 7 belong in other August notebooks)
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[[Team:University_of_Lethbridge/Notebook|Back to The University of Lethbridge Main Notebook]]
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===August 1, 2008===
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====Nathan Puhl, Roxanne====
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Riboswitch 20 uL PCR. Set up 4 reactions (25 uL for each total volume). 1 uL or 1/100 pTopp and 1 uL or H1/100 PCR from July 28, 2008.
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PCR conditions:
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A. Initial denaturation: 98 C (3 min)
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B. -Denaturation: 98 C (10 sec)
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    - Annealing: 55 C (30 sec)
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    -Extension: 72 C (15 sec)
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    -30 cycles
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C. Final extension: 72 C (7 min)
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===August 2, 2008===
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====Nathan Puhl, Roxanne====
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Ran riboswitch (Aug. 1, 2008) on 3% agarose gel. Results are in the hard copy lab notebook. Looks like 76 bp band will extract.
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===August 5, 2008===
===August 5, 2008===
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Gel extracted LacI insert and DT vector. Didn't run gel yet.
Gel extracted LacI insert and DT vector. Didn't run gel yet.
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===August 7, 2008===
 
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====Nathan Puhl, Roxanne====
 
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Riboswitch:
 
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Set up PCR using purified riboswitch from Aug. 5, 2008 with platinum Taq (50 uL reaction). Made Master Mix for three reactions.
 
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Master Mix:
 
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-10x Buffer (no Mg2+): 15 uL
 
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-10 mM dNTPs: 3 uL
 
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-50 mM Mg2+: 4.5 uL
 
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-10uM RF: 3 uL
 
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-10uM RR: 3 uL
 
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-Plat. poly: 0.6 iL
 
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-H20: 120.9 uL
 
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-template: 1 uL
 
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Cycle conditions:
 
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A. Initial denaturation: 94 C (2 min)
 
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B. -Denaturation: 94 C (30 sec)
 
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    - Annealing: 55 C (30 sec)
 
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    -Extension: 72 C (30 sec)
 
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    -30 cycles
 
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C. Final extension: 72 C (7 min)
 
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====Roxanne====
 
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-Ran the PCR Product on a 3% gel using only 1uL of DNA from the riboswitch.
 
===August 13, 2008===
===August 13, 2008===
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Ran Plasmids in a 1% Agarose Gel at 100 V for 30 minutes.
Ran Plasmids in a 1% Agarose Gel at 100 V for 30 minutes.
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===August 16, 2008===
 
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====Nathan Puhl, Roxanne, Munima====
 
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-Restriction Digested the purified riboswitch and pSB1A7 with XbaI and SpeI, let it run for 4 hours
 
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====Nathan Puhl, Roxanne====
 
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-Ran all of the restricted pSB1A7 plasmid through a 1% agarose gel at 100V for 25 minutes.
 
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-Ran a gel extraction on the pSB1A7 cut plasmid, and ran a PCR clean-up reaction on the digested RS1 and RS2 amplicons.
 
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-Ran 1 uL of each on a 1% to quantify the amount of DNA present.
 
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-Ligated RS1 + pSB1A7, and RS2 + pSB1A7, using T4 DNA Ligase.
 
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-1 uL of RS1/RS2
 
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-4 uL of pSB1A7
 
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-1 uL of 10x T4 DNA Ligase Buffer
 
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-0.33 uL of T4 DNA Ligase
 
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-3.67 uL of water
 
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allowed the reaction to go overnight
 
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===August 17, 2008===
 
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====Nathan Puhl====
 
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-Transformed DH5a cells with the pSB1A7 + RS1, and pSB1A7 + RS2 plasmids.
 
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-Plated on semi-solid agar plates containing 100 ug/mL of ampicillin.
 
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===August 18===
 
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====Christa, Nathan Puhl, Munima====
 
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-Nathan checked the plates for growth. Colonies are present.
 
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-Ran a colony PCR of the pSB1A7 + RS1, and pSB1A7 + RS2 recombinant cells transformed by Nathan and Roxanne on August 17.
 
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-Inoculated the cells into tubes of liquid media + 100 ug/ml of ampicillin.
 
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Roxanne will remove the PCR tube from the thermocycler in the morning.
 
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===August 19, 2008===
 
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====Roxanne====
 
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-Ran the PCR Product on a 2% Agarose Gel at 100 V for 33 minutes.
 
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[[Image:RS1 RS2 gel.jpg| 350 px]]
 
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-Plasmid Prepped and made glycerol stocks from the RS1-1 and RS2-1 tubes of cells incubated in LB media + 100 ug/mL ampicillin.
 
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Ran the pRS1 and pRS2 plasmids on a 1% Agarose Gel at 100 V for 30 minutes.
 
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[[Image:pRS1 pRS2 gel.jpg| 150 px]]
 
====Christa, Munima, Sebastian====
====Christa, Munima, Sebastian====
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Conclusion: The bands appeared to be at the correct size for pSB1A7.
Conclusion: The bands appeared to be at the correct size for pSB1A7.
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===August 21===
 
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====Nathan Puhl, Roxanne====
 
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-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.
 
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===August 21, 2008===
 
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====Roxanne====
 
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-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.
 
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====Nathan Puhl, Roxanne====
 
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-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.
 
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-setup a restriction digest for pSB1A7 using XbaI and SpeI, ran overnight.
 
===August 22, 2008===
===August 22, 2008===
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   and CheZ were estimated to be 25 ng/uL and 80 ng/uL, respectively.
   and CheZ were estimated to be 25 ng/uL and 80 ng/uL, respectively.
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Next step: Digest pSB1A7 with alkaline phosphatase. A restriction digest will be performed at a later date on CheZ to prepare it for insertion.
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Next step: Digest pSB1A7 with antarctic phosphatase. A restriction digest will be performed at a later date on CheZ to prepare it for insertion.
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===August 23, 2008===
 
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====Nathan Puhl, Roxanne====
 
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-Digested pSB1A7 with Antartic Phosphatase
 
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  -9 uL of cut pSB1A7
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===August 26, 2008===
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  -1.5 uL of 10x Antarctic Phosphatase Buffer
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====Roxanne, John====
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  -1 uL of Antarctic Phosphatase Enzyme
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-Restriction Digested the Biobrick Parts I13504, I13401, P0440, C0014, B0015, J31007 with XbaI and PstI
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  -3.5 uL of water
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  -20 uL template (~2 ug)
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  Allowed the Reaction to take place for 30 minutes to remove the 5` Phosphates from the pSB1A7 plasmid to
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  -5 uL React 2
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  prevent religation.
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  -4 uL XbaI
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  -4 uL PstI
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  -17 uL ddH2O
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  ____
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50uL Reaction in 37.0C H2O bath overnight
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===August 27, 2008===
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====Roxanne====
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-Inactivated the Restriction Enzymes by placing them on the heating block at 65.0C for 10 minutes
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-Ran 25uL of DNA on a 1% Agarose Gel at 100 V for 30 minutes
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-Gel Extracted the DNA
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-Ran 1 uL of DNA in a 1% Agarose Gel at 100V for 30 minutes.
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[[Image:Biobrick_Parts.jpg| 250 px]]
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===August 28, 2008===
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====Roxanne, Munima, Sebastian, Nathan Puhl====
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-Performed a restriction digest on LacI+dt, pLacI, pStrong, and RFP sub
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 +
-10 uL template
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-5 uL NEB 2
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  -2 uL Restriction Enzyme #1 (Xba I or Spe I)
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-2 uL Restriction Enzyme #2 (Pst I)
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-21 uL ddH20
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____
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50 uL Rxn left to run overnight at 37.0
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-Ran the remainder of the pSB1A7 plasmid from August 22nd on 1 1% Agarose Gel at 100 v for 27 minutes.
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-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.
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-Gel Extracted the plasmid DNA.
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-Picked a colony from the dT plate which was stored in the fridge from several weeks ago, incubated overnight at 37.0C
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-Purified the Phosphatase reaction to isolate the pSB1A7 DNA.
 
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-Ran a 1% gel to quantify the amount of plasmid DNA present.
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===August 29, 2008===
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====Roxanne, Nathan Puhl, Munima, Sebastian, Andrew====
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-Performed the ligation of the riboswitch into pGEM T-easy, transformed and plated.
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-Ligated RS1 and RS2 into the dephosphorylated pSB1A7 using T4 DNA Ligase.
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=====Roxanne====
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-Gel Extracted the remainder of the DNA
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-1 uL of RS1 or RS2
 
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-4 uL of dephosphorylated pSB1A7
 
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-1 uL of 10X T4 DNA Ligase Buffer
 
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-0.33 uL T4 DNA Ligase Enzyme
 
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-3.67 uL water
 
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Reaction was allowed to go overnight
 
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===August 24, 2008===
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===August 30, 2008===
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====Nathan Puhl====
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====Nathan Puhl, Roxanne, Andrew====
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-Transformed DH5a cells with the RS1+pSB1A7 or RS2+pSB1A7 plasmid on semi-solid agar plates containing 100 ug/mLof amppicillin.
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-Ran a gel of the Digested Parts on 1% Agarose @ 100V for 27 minutes.

Latest revision as of 02:29, 30 October 2008

Back to The University of Lethbridge Main Notebook

Contents

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 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 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.

Biobrick Parts.jpg


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

-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

-Ran a gel of the Digested Parts on 1% Agarose @ 100V for 27 minutes.

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