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===Procedure=== ====First Strand Synthesis==== {| {{table}} | align="center" style="background:#f0f0f0;"|'''Component''' | align="center" style="background:#f0f0f0;"|'''Pos Ctrl''' | align="center" style="background:#f0f0f0;"|'''Neg Ctrl''' | align="center" style="background:#f0f0f0;"|'''Exp''' |- | 25mM dNTP||1||1||1 |- | 1.3ug/ul UHRR||0.5||0.5||0.5 |- | 100uM RanHex Primer||0.5||0.5||0 |- | 100uM Top48 Primer||0||0||0.5 |- | H2O||7||7||7 |} #Heat reaction mix (above) to 65C for 5min #Add 1ul 10X M-MuLV RT Buffer (total 10ul volume) #Incubate 25C for 10min #Add 1ul M-MuLV Reverse Transcriptase to Pos Ctrl and Exp ONLY (1ul H2O for Neg Ctrl) #Incubate 42C for 60min #Inactivate by heating to 85C for 10min ====Second Strand Synthesis==== #Add 57ul H2O #Add 8ul 10X Second Strand Synthesis Buffer #Add 4ul Second Strand Enzyme #Mix by pipetting #Incubate 16C for 2.5 hours #Purify with magnetic bead (120ul beads to 80ul reaction) #*Elute with 25ul H2O ====Gel Check==== *2ul sample + 2ul 6X loading dye *0.5ul Low Mass Lader + 2ul H2O + 2ul 6X loading dye *First 2 gels ripped because stuck to the walls of the cassette [[File:2014-06-10_RTPrimerinvitroTest.jpg |250px]] *Positive and negative control look the same **Talking to Andrew, he pointed out that gels can't differentiate between single stranded, double stranded, and DNA vs RNA ***Possibly continue with Nextera since that will definitely tell us whether it worked or not Continued on: [[Matt:LabNotes/2014-6-13]] ====Qubit Quant==== *PosCtrl: 1.06ng/ul *NegCtrl:0.884ng/ul *Exp:0.818ng/ul
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