Team:BYU Provo/Notebook/Cholera - Enzyme/September/Period1/Dailylog

Overview

March-April

May-June

July-August

September

1. 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.
2. 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.
3. 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.
4. 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.
5. 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.
6. 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.
7. 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

1. Cro PCR
2. Qrr4/RFP PCR
3. piG91 miniprep

As well 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

9/5/2013

I ran the PCR products from 9/4 on gel. There were no viable products.

9/6/2013

We set up PCRs with Desi with a variety of conditions to test all of the primers that we have been using and the new primers for the iGem plasmid backbone for both DspB and Savinase. This will allow us to see if the DNA templates that we are using are viable or not. If some of the PCRs work, but not all of them, then we know that the problem is with our primers, not the DNA templates we are using.

9/9/2013

We ran our PCR products from 9/6 on gel and got no results on any of them. This clearly shows that our DNA templates themselves are not viable. We will need to get new DNA templates to use for DspB and Savinase

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/MJS