User:Anthony Salvagno/Notebook/Research/2010/02/01/Jasper PCR improvement attempts

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Email to Osley

I’m trying t do a 100bp PCR with Cy3/Cy5 labeled 5’ ends (on the primers) and I haven’t been able to get it to work over here. The belief is that our Thermal cycler may not be good enough for the precision of this reaction. Would it be possible to come in late today or late tomorrow to use your Thermal Cycler for the reaction? Doing it late would allow me to not disturb your guys usage of it and to run it over night when I could get it early the next day. If this is possible could you schedule me on your time sheet and let me know when it is available for this use? This is of course if you will permit me the access in the first place. Thanks guys and I hope to see you soon.

PCR Protocols of 100bp product


SJK 20:33, 1 February 2010 (EST)

20:33, 1 February 2010 (EST)
Good article finds and good email to Osley. Did you get a response?

_fmt=high&_orig=search&_sort=d&_docanchor=&view=c&_acct=C000053660&_version=1&_urlVersion=0&_userid=1550512&md5=cb1dfb8e92551a8ba19ed7979bf34c0a#secx2 Identification of mammal species using species-specific DNA pyrosequencing]=== They say:

The same PCR amplification conditions were used for both the 12S rRNA and the 16S rRNA sequences: 1× PCR buffer II (Applied Biosystems), 3.0 mM MgCl2, 100 μM each of dATP, dCTP, dGTP, 200 μM of dUTP, 0.016% BSA, 2% glycerol, 200 nM of each primer (Table 2), 0.05 U of uracil–DNA glycosylase (USB corporation), 1.25 U of AmpliTaq Gold DNA polymerase (Applied Biosystems) and 5 μl template DNA in a total volume of 25 μl. The amplification programme was: 10 min at 37 °C, 5 min at 95 °C followed by 35 cycles of 30 s at 94 °C, 30 s at 57 °C, 30 s at 72 °C with a final extension for 10 min at 72 °C and was conducted in a GeneAmp® PCR System 9700

I have thought about lowering the melting temp to 55C because many sources say that is a good place to start. I don't know though.

Guest, a transposable element belonging to the Tc1/mariner superfamily is an ancient invader of Neurospora genomes

They say:

PCR amplifications (Saiki et al., 1988) used a reaction mixture comprising: 200 mM dNTPs (Ameresco), 1x reaction buffer (Perkin–Elmer), 2.5 mM MgCl2, 400 ng of the single primer GTACACATCAACCCTGGT (Geneworks) based on the Guest TIR, 1.25 units AmpliTaq Gold DNA polymerase (Perkin–Elmer) and 400 ng DNA in a total reaction volume of 50 μl. Hotstart thermal cycling was 94 °C, 1 min followed by 24 cycles of 94 °C, 1 min; 65 °C, 1 min; 72 °C, 3 min with a 1 °C decrement in annealing every cycle; then 10 cycles of 94 °C, 1 min; 50 °C, 1 min; 72 °C, 3 min; and finally 72 °C, 7 min before finishing at 4 °C. A Gene Amp 2400 (Perkin–Elmer) was used for all thermal cycling.

They do some decrement technique where they lower the annealing temp each time for 24 cycles then just raise it to 50C. I don't know what that benefit is and they don't cite anything that talks about that technique. Biology is a word of mouth science.