Team:Baltimore US/SeptGroupNotebook

Group Notebook for September 2010

Sept 30

@ Burkett Lab Amplify more template -Robert received large bands on gel. Will cut out and purify on Tuesday

Sept 28

@ Burkett Lab Robert ran gels on Prefix & Suffix. Prefix did not come out well, Suffix fine.
Remixed primers, tested templates/all parts for contamination -zip
After further overlap investigation, Robert discovered that several changes were necessary
to see successful overlap results. 1st: Our template should be 5X the amount we had been using
2nd: Pfu Turbo is required as opposed to the Pfu we had been using.
3rd: We are to add the Primers After Cycle 15 during PCR

Sept 27

@ Burkett Lab Bernadette & Ryan
Spectrometer readings on 2 samples Prefix & Suffix were as follows:
P1 Absorb = .818 Concen =40.8863 ug/ml P2 Absorb = .771 Concen. = 38.5439 ug/ml
S1 & S2 combined for larger sample Absorb = 1.221 Concen = 61.0552 ug/ml
Overlap pcr attempt #5.. Annealing 59 degrees C, Melt 95 degrees, Extension 72
Same basic results as before, checking the template quality

Sept 23

@ Burkett Lab Bernadette's notes
Robert did PCR for Suffix with Alicia's assistance. Robert purified the Suffix & Bernadette purified
the Prefix. In freezer with iGEM label P1 & P2 for prefix and S1 & S2 for Suffix
New Pfusion 400 enzyme arrived -prepared for overlap Monday!

Sept 21

@ Burkett Lab Bernadette & Robert present
Bernadette's notes
Overlap extension problems have taken up several labs. Repeated PCR results have not established
overlap success. Reran at different temps/times, confirmed integrity of materials, etc
After further research on the overlap process, Robert brought to our attention a critical template
volume error. In addition, primers are supposed to be added during the PCR reaction
and a different Pfusion variant should be used. We are now prepping for the next phase.

Yesterday's lab, I prepared prefix & suffix primers for PCR to replenish our supply.
5 Prefix reactions, labeled #1 thru 5: diH20 25.5 ul, buffer 10ul, dNTP 1 ul, DMSO 2.5 ul,
2.5 ul of FW primer & 2.5 ul Taq RM primer, 5 ul Taq DNA, 1 ul Pfu
5 Suffix reactions, labeled 6-10,identical to the 1st 5 reactions except the suffix primers were used
Taq FM 2.5 ul and RV 2.5 ul. I also ran a template control labeled 11 and a primer control labeled 12

Hot started PCR at 98 degrees C for 1 minute - paused, added Pfu, PCR ranging from 57-98 for 35 cycles
Today, I ran the gels for the reactions. Suffix problem (labeled 6-10) that I'll rerun Thursday.
Prefix looked good. I cut out the gel for purification, also to be completed Thursday.

Sept 9

@ Burkett Lab Robert & Bernadette present
Bernadette's notes:
Robert ran gel from overlap with not the results expected - faded bands & not the correct size
We discovered that overlap PCR not run at optimum annealing temp of 59 degree C where we
previously had success.
We prepped 4 more experiments - 2 as controls.
Bernadette prepped #1: diH20 31 ul, buffer 10 ul, dNTPs 1 ul, Fw Primer 2.5 ul, Rv Primer 2.5 ul,
DNA Prefix 1 ul, Dna Suffix 1 ul. Hot Shot start @ 98 degrees before adding enzyme, 1 ul Pfu
Bernadette prepped for Control PCR #4, identical to #1 but no DNA & 33 ul diH20
Robert prepped #2, identical to #1, except doubled DNA to 2 ul each & 29 ul diH20
Robert also prepped Control PCR #3 with no primers & 36 ul diH20.

We are very optimistic that our PCR gels will confirm our expected overlap results Monday

Sept 7

@ Burkett Lab Robert, Ryan & Bernadette present

Previously cut DNA from gels. Ryan & Robert performed extraction with a 'Spin Gel Extraction Kit'
Spectrometer readings confirmed we have samples!
Prefix = 1.777 absorption w/concentration 88.8718 ug/ml of sample
Suffix = 1.762 absorption w/concentration 88.0820 ug/ml of sample

PCR prepped for overlap