Wednesday, July 22, 2015

Sanguine PCR of a PCR

Objective: Taking the product of the PCR run on 15, july 2015 and running it out on a Gel to see if the PCR of the PCR product from 1, july 2015 was successful. Hopefully it was amplified enough to be able to sequence. Sam is gone for the week so I will be able to get verify next week.

50ml 1x TBE
.5 grams agarose (1%)
5 ul athidium bromide
Gel run with ogene ruler 100 bp dna ladder (thermo scintific #sm1143 lot # 00189314)
(PCR product 1 and 2 were taken from the combining of two gel bands from previous PCR)

1 5ul Ladder
2 PCR product 1
3 PCR Product 2
4 H2O control 1
5 H2O control 2

I was also having trouble interpreting the results from the Muscle Alignment and so I talked with Jake who recommended some names (DR. Carrie Naish, Charlie Waters, and the Seebs) to try to get in contact with that have more experience with Geneious.

175B MAR
175A MAR
209 MAR


The PCR was successful the bands in row 1 and 2 that had the combined PCR product from the previous PCR were amplified correctly and show up much brighter than the previous. I removed the bright bands from the 1000 bp mark on the ladder and put them in separate tubes. I noticed a mistake that I had been using another ladder diagram that shows the bands at 1000bp which was incorrect the correct is shown on this I will replace the images on the others with the correct ladder. i still show that the sequencing that we have been getting is still half of what is being sent not likely reason why the length is so much smaller. 500 bp is still smaller than the expected ~850 that I am looking for. Supports the idea that new primers may be needed. All in all a nice looking PCR I believe but will verify later and hopefully get this run off for sequencing next week. Jake brought up that the bands were extremely close together so without good separation that it would be difficult to know that the bands that I cut out are the strands that I am looking for. When the bands are so close together it is very easy to get "junk" other than what I want. This may mean that sequencing may not be an option.  also noticed that when taking a picture with my phone it was easier to take the orange filter and lay it on the cover of the machine. My phone seemed to focus a lot quicker and took a better picture then just holding the filter directly on the camera phone.

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