Team:University of Lethbridge/Notebook/GeneralLabAugust

From 2008.igem.org

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===Augest 13, 2008===
+
[[Team:University_of_Lethbridge/Notebook|Back to The University of Lethbridge Main Notebook]]
-
====Nathun Puhl and Sebastian====
+
===August 5, 2008===
-
Varified and quantified the LacI and DT restriction digest products.
+
====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
Set up a Ligase experiment for LacI and DT
Line 11: Line 42:
  - 7.2 uL ddH2O
  - 7.2 uL ddH2O
  - 20 uL total volume
  - 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.
 +
 +
[[Image:Biobrick_Parts.jpg| 250 px]]
 +
 +
 +
===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.

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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