|
9/4/13
Today we set up new overnights of cholera by adding 4 mL of SLB and 50 uL of our previous overnight from 8/26/13 into a test tubes and placing in the 30° incubator overnight. We prepared two overnight cultures and one control.
We also ran the GenElute Plasmid Miniprep Kit on the recombinant E. coli cells containing the piG91 plasmid using the protocol below to pull the iGem plasmid backbone out of the E. coli cells in preparation to clone our biobrick parts into the iGem plasmid backbone for submission to the iGem registry. We prepared two minipreps and the final product concentrations were: 28.1 ng/uL and 36.0 ng/uL.
Protocol:
- Pellet 1-5 mL of overnight culture of recombinant E. coli culture by centrifugation at ≥ 12,000 × g for one minute. Resuspend pellet in 200 uL of provided Resuspension Solution.
- Lyse resuspended cells by adding 200 uL of Lysis solution. Immediately mix contents by gentle inversion 6-8 times, until the mixture becomes clear and viscous. Allow the Lysis reaction to proceed for approximately two minutes, but do not exceed five minutes.
- Neutralize lysis and precipitate cell debris by adding 350 uL of Neutralization/Binding solution. Gently invert 4-6 times. Pellet cell debris by centrifuging at ≥ 12,000 × g for 10 minutes. Cell debris will precipitate out.
- Insert a GenElute Miniprep Binding Column into a microcentrifuge tube. Add 500 uL of Column Preparation Solution and centrifuge at ≥ 12,000 × g for 30 seconds to 1 minute; discard flow through liquid.
- Transfer clear lysate from step 3 to column from step 4 and centrifuge at ≥ 12,000 × g for 30 seconds to 1 minute; discard flow through liquid.
- Add 750 uL of the diluted wash solution to the column. Centrifuge at ≥ 12,000 × g for 30 seconds to 1 minute. Discard flow through liquid and centrifuge again for 1 to 2 minutes, to remove excess ethanol.
- Transfer column to a fresh collection tube. Add 50 uL of Elution Solution to the column and let stand for 5 minutes.
We then set up Restriction Digests for the Cro PCR, Qrr4/RFP PCR, and piG91 mini prep products using the setup shown below.
Restriction Digest setup
7.5 uL H2O
4.0 uL Buffer 4
4.0 uL 10x BSA
20 uL DNA template
1.5 uL Xba 1
1.5 uL Spe 1
Digests Labelled
- Cro PCR
- Qrr4/RFP PCR
- piG91 miniprep
-MJS
Today we set up phusion and Taq PCR for B. subtilis and phusion PCR for dspB
Phusion PCR setup
35 microliters ddH2O
10 microliters 5X phusion buffer
1.5 microliters 10mM dNTP's
1 microliter each primer
1 microliter diluted template DNA
.5 microliter phusion polymerase
Taq PCR setup
40 microliters ddH2O
5 microliters 10X TAQ buffer
1.5 microliters 10mM dNTP's
1 microliter each primer
1 microliter diluted template DNA
.5 microliter phusion polymerase
-NRS
9/5/2013
I ran the PCR products from 9/4 on gel. There were no viable products. -NRS
9/6/2013
Dezi helped us set up the PCR again using the primers to clone into the iGEM plasmid to see if our other primers are the problem.
-MJS/NRS
9/9/2013
We set up the new cholera overnights. The gel for the PCR products showed no viable results for anything. We found a new source of DspB which should be here Wednesday as well as another sample of B. subtilis so we can re-start from those. -NRS
| 
"
