User:Anthony Salvagno/Notebook/Research/2010/01/26/Jasper PCR Try 4 and Testing Protocol
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Try 4.1
{{#widget:Google Spreadsheet |key=tGKpNt5Z7jCOknOGU0aq1sw |width=500 |height=300 }} As you can see, I am diagnosing potential problems with the reaction by running the 1.1kb reaction. I am also running the Cy3 primer reaction just for shits and giggles. Not shown is an extra reaction labeled with a "?" using the same components as tubes 1 and 2, but from different tubes (ie the 10x buffer in both are the same in that they are both from invitrogen and are 10x PCR buffer -MgCl2, but they are not from the same tube of 10x PCR buffer). Koch will run the gel analysis for me since I will be in class. Picture to come when gel is done.
Gel Analysis
I don't know why this camera took such a shitty image, but as per Koch's loadings, the lane designations are:
- 100bp ladder
- tube 1 - 1.1kb PCR
- tube 2 - 1.1kb PCR
- 100bp ladder
- tube 3 - Cy3 Jasper PCR
- tube ? - 1.1kb PCR from different tubes
Strangely tubes 1 and 2 didn't work right, tube ? did work well and I got product in tube 3. Hopefully the second PCR for the day will work well and I can finally purify some product and give to collaborator.
Note how bright the primer for the Cy3 PCR is. I just calculated that 10uM of primer is around 166ng/ul and I have 20uM of both primers (combined). I'm pretty sure the brightness of the band doesn't equate to the mass of the DNA (based on prior experience) and definitely think some of the excitation belongs to the Cy3 tags. Pretty cool IMHO.
Try 4.2
Since the earlier PCR worked (finally!) I will do both products trying again from try 1.1 but with more primer mix, thus try 4.2 is here: {{#widget:Google Spreadsheet |key=tO90T464n5cXV_nGss3e7Vw |width=500 |height=300 }} Gel results to come tomorrow.
