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===results=== *T7-top3 had satisfactory fragmentation profile. Will try perform IVT, polyA tailing, RT, and PCR and see if quality smear is obtained. *If quality smear obtained, then size selection, cloning and transformation for validation. Use blunt end kit for to ensure ligations into vector. [[File:ZhangLab 2 2012-05-31 11hr 24min-labeled.jpg|600px]] *T7-top3 RNA qualitative results with TBU gel *odd that needed 3 uL of sample to confirm presence of smear (barely visible with 1 uL sample) *assume that taq polymerase is going bad since have freeze thawed many time in the past few weeks, will start using a new aliquot. Otherwise controls look fine. Also sample 8 is 1 uL of T7tspn-top2 from a previous IVT and has nice smear.. have lots of RNA to use for experimentation. [[File:ZhangLab 2 2012-05-31 18hr 11min-labeled.jpg|600px]] *TBE gel after polyA tailing, RT, PCR *T7tspn-top3 for first 7 lanes. lane 8 is new primers on T7-tspn-top2 *backgound probably due to tranposon amplification? control looks like top3 custom transposon sample [[File:ZhangLab 2 2012-06-04 17hr 55min-labeled.jpg|600px]] *TBU gel after polyA tailing, RT, PCR *Looks like no large products amplified, is all junk. *Will try fragmentation methods and then polyA tailing, or could try denaturation [[File:ZhangLab 2 2012-06-04 19hr 24min-labeled.jpg|600px]]
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