Difference between revisions of "Dustin Atchley"
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Don't want full length DNAs, because we want about 75 bases for short reads. | Don't want full length DNAs, because we want about 75 bases for short reads. | ||
Randomly fragmented which is wonderful. | Randomly fragmented which is wonderful. | ||
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| + | Gel purified and reamplified (picture in PP) | ||
Revision as of 19:53, 12 January 2016
Hello world!
Most common mistake "I will remember how I got here". Take good NOTES!
Harvest .1 g of tissue, 100 micrograms of RNA from RNA isolation kit. Don't know we didn't sample the wrong part of the organ since the amount is so small. Be your hardest critic.
Isolated PolyA RNA (Ideally you get 2% of the RNA, protein coding)
Fragment the RNA before synthesizing.
DNA polymerase (reverse transcriptase) goes into mix, made cDNA.
Primers for amplification, you make a hexamer, which is every possible combination so that the complementary bases will bind.
Sequence cDNA that is the template to the RNA you are using. It is more stable.
Don't want full length DNAs, because we want about 75 bases for short reads.
Randomly fragmented which is wonderful.
Gel purified and reamplified (picture in PP)
