Team:University of Lethbridge/Notebook/GeneralLabAugust

From 2008.igem.org

(Difference between revisions)
m (Christa, Nathan Puhl, Munima)
m (Nathan Puhl)
Line 123: Line 123:
====Nathan Puhl====
====Nathan Puhl====
-Transformed DH5a cells with the pSB1A7 + RS1, and pSB1A7 + RS2 plasmids.
-Transformed DH5a cells with the pSB1A7 + RS1, and pSB1A7 + RS2 plasmids.
 +
-Plated on semi-solid agar plates containing 100 ug/mL of ampicillin.
-Plated on semi-solid agar plates containing 100 ug/mL of ampicillin.

Revision as of 22:08, 24 August 2008

(Aug. 1- 7 belong in other August notebooks)

Contents

August 1, 2008

Nathan Puhl, Roxanne

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.

PCR conditions:

A. Initial denaturation: 98 C (3 min)
B. -Denaturation: 98 C (10 sec)
    - Annealing: 55 C (30 sec)
    -Extension: 72 C (15 sec)
    -30 cycles
C. Final extension: 72 C (7 min)

August 2, 2008

Nathan Puhl, Roxanne

Ran riboswitch (Aug. 1, 2008) on 3% agarose gel. Results are in the hard copy lab notebook. Looks like 76 bp band will extract.

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 7, 2008

Nathan Puhl, Roxanne

Riboswitch:

Set up PCR using purified riboswitch from Aug. 5, 2008 with platinum Taq (50 uL reaction). Made Master Mix for three reactions. Master Mix:

-10x Buffer (no Mg2+): 15 uL
-10 mM dNTPs: 3 uL
-50 mM Mg2+: 4.5 uL
-10uM RF: 3 uL
-10uM RR: 3 uL
-Plat. poly: 0.6 iL
-H20: 120.9 uL
-template: 1 uL

Cycle conditions:

A. Initial denaturation: 94 C (2 min)
B. -Denaturation: 94 C (30 sec)
    - Annealing: 55 C (30 sec)
    -Extension: 72 C (30 sec)
    -30 cycles
C. Final extension: 72 C (7 min)

Roxanne

-Ran the PCR Product on a 3% gel using only 1uL of DNA from the riboswitch.

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.

August 16, 2008

Nathan Puhl, Roxanne, Munima

-Restriction Digested the purified riboswitch and pSB1A7 with XbaI and SpeI, let it run for 4 hours


Nathan Puhl, Roxanne

-Ran all of the restricted pSB1A7 plasmid through a 1% agarose gel at 100V for 25 minutes. -Ran a gel extraction on the pSB1A7 cut plasmid, and ran a PCR clean-up reaction on the digested RS1 and RS2 amplicons. -Ran 1 uL of each on a 1% to quantify the amount of DNA present. -Ligated RS1 + pSB1A7, and RS2 + pSB1A7, using T4 DNA Ligase.

-1 uL of RS1/RS2
-4 uL of pSB1A7
-1 uL of 10x T4 DNA Ligase Buffer
-0.33 uL of T4 DNA Ligase
-3.67 uL of water

allowed the reaction to go overnight


August 17, 2008

Nathan Puhl

-Transformed DH5a cells with the pSB1A7 + RS1, and pSB1A7 + RS2 plasmids.

-Plated on semi-solid agar plates containing 100 ug/mL of ampicillin.

August 18

Christa, Nathan Puhl, Munima

-Nathan checked the plates for growth. Colonies are present.

-Ran a colony PCR of the pSB1A7 + RS1, and pSB1A7 + RS2 recombinant cells transformed by Nathan and Roxanne on August 17.

-Inoculated the cells into tubes of liquid media + 100 ug/ml of ampicillin.

Roxanne will remove the PCR tube from the thermocycler in the morning.

August 19, 2008

Roxanne

-Ran the PCR Product on a 2% Agarose Gel at 100 V for 33 minutes.

RS1 RS2 gel.jpg

-Plasmid Prepped and made glycerol stocks from the RS1-1 and RS2-1 tubes of cells incubated in LB media + 100 ug/mL ampicillin.

Ran the pRS1 and pRS2 plasmids on a 1% Agarose Gel at 100 V for 30 minutes.

PRS1 pRS2 gel.jpg

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 digested pSB1A7 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 both pSB1A7 and CheZ with phosphatase. A restriction digest will be performed at a later date on CheZ to prepare it for insertion.