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== Amplification == {| {{table}} border = 1 | align="center" style="width:250px;background:#f0f0f0;"|'''Components''' | align="center" style="width:80px;background:#f0f0f0;"|'''1X 50ul rxn''' | align="center" style="width:80px;background:#f0f0f0;"|'''10ul rxn''' | align="center" style="width:80px;background:#f0f0f0;"|'''6 rxn''' |- | Adaptor ligated DNA||10.00||2.00||0.00 |- | 2X KAPA HiFi Hotstart ReadyMix||25.00||5.00||30.00 |- | 10uM TruS F/R or 10uM SG_PCR F/RInd1||1.50||0.30||0.00 |- | 50X SYBR||0.40||0.08||0.48 |- | H2O||13.10||2.62||15.72 |- | Total ||50.00||10.00|| |} * I split PCR mix into two tube and add primers for TruSeq adaptor (add 0.6ul of 10uM TruS F/R to 15.4ul of PCR mix and add 1.2ul of 10uM SG_PCR F/RInd1 to the left over PCR mix in the tube) :- Aliquot 8 + 2ul adaptor ligated DNA :- Mix well ::''98C for 45sec --> [98C for 15sec -> 60C for 30sec -> 72C for 30sec] X 9 --> 72C for 1min'' * I stopped at 9 cycles to avoid amplification saturation for a precise comparison. [[File:2014-11-28_qPCR-vary-customAdapt-9X_300nMprimer.PNG| 550px]] * This time, the qPCR curves of 1X TruSeq and 1X customized adaptor were pretty much the same which is different from previous experiment on 2014-11-25. I will look at gel image for comparison with qPCR curves. :- Loaded 3ul of PCR product in 6% TBE gel [[File:ZhangLab_2 2014-11-28 19hr 20min_vary-custAdaptor_9X.jpg| 300px]] :- I also did PAGE quantification to compare the yield of each condition ==== PAGE quantification result ==== '''Adaptor Relative intensity to TruSeq 1X (14nM)''' TruS_1X 1.00 UMI_1X 0.71 UMI_2X 0.87 UMI_4X 1.05 * From this result, Dr. Zhang suggested that it should be fine to use 2X of adaptor (28nM) in ligation reaction.
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