Matt:LabNotes/2014-6-13
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Top48 Hexamer RT Primer in vitro Validation: Nextera Tagmentation[edit]
- Continued from: Matt:LabNotes/2014-6-9
- Since running a gel without digesting RNA (eg with RNase A) can't confirm success of RT, must continue with Nextera, and successful tagmentation indicates successful RT and Second Strand Synthesis
Protocol[edit]
- Taken from Andrew's protocol
Tagmentation[edit]
- Add 2 ul HMW buffer to each PCR tube
- Transfer 7ul of each sample to tubes
- PosCtrl: 1.06ng/ul
- NegCtrl: 0.884ng/ul
- Experimental: 0.818ng/ul
- NexteraControl (gDNA): 0.803ng/ul
- Add 1 ul transposase (1:50 dilution) to each sample. Incubated 5.5 minutes at 55 degC
- Dilute Qiagen protease 1:100 in H2O. Add 1 ul to each sample and incubate 10 minutes at 50 degC, followed by 20 minutes at 70 degC
- Make the master mix for Exo- treatment: 1 ul Exo- and 0.4 ul dNTP per reaction
- Add 1.4 ul Exo- mix to each sample and incubate 15 minutes at 37 degC, followed by 20 minutes at 75 degC
PCR with adapters and primers[edit]
- 6-17-2014
- Finish first round PCR master mix (per reaction):
- 15 ul 2X KAPA fast SYBR.
- 0.6 ul Adapter 1.
- 1.4 ul H2O.
- Add 0.6 of barcode adapters to each sample.
- PosCtrl: Indx 26
- NegCtrl: Indx 27
- Exp: Indx 28
- gDNA: Indx 29
- Add 17 ul of first round PCR master mix to each sample.
- Incubate in thermocycler:
- 95 degC for 1 min.
- 7 cycles:
- 95 degC for 10 sec.
- 62 degC for 15 sec.
- 72 degC for 45 sec.
- Finish second round PCR master mix:
- 35 ul KAPA fast SYBR
- 2 ul Primer 1
- 2 ul Primer 2
- 31 ul H2O
- Add 70 ul of second round PCR master mix to each sample.
- PCR program is as follows:
- 95 degC for 1 min.
- 10 cycles:
- 95 degC for 10 sec.
- 60 degC for 15 sec.
- 72 degC for 45 sec.
- Incubate in thermocycler an additional 3 min @ 72 degC for final extension.
File:2014-06-17 Top48HexPrimer NexteraPCR.jpg
- Run 5 ul sample on PAGE gel with 3 ul 6X loading dye to identify positives.
- 0.5 ul low mass ladder (Invitrogen). Equalize volume with TBE.
- 25 min @ 250 V.
- Stain 5-10 min in ~100 ml 1X TBE with 6 ul SYBR Safe.
File:2014-06-17 RTPrimerinvitroTest Nextera.jpg
Conclusion[edit]
- Negative Control had double stranded DNA somehow nullifying the experiment