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=== 7) PCR amplification === ==== <u>1st round PCR</u> ==== '''''<span style="color:crimson"><u>Prep</u></span>'''''<br> * I used all eluted bisDNA in the 1st round PCR :{| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="width:80px;background:#f0f0f0;"|'''Conc''' | align="center" style="width:80px;background:#f0f0f0;"|'''unit''' | align="center" style="width:80px;background:#f0f0f0;"|'''Final conc.''' | align="center" style="width:80px;background:#f0f0f0;"|'''unit''' | align="center" style="width:80px;width:80px;background:#f0f0f0;"|'''Volume (ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''50 rxn mix''' |- | Bis-cvt DNA||||||||||30.00||0.00 |- | 10X Reaction buffer||10||X||1||X||5.00||250.00 |- | dNTP mix||10||mM||0.3||mM||1.50||75.00 |- | TruF/R||10||uM||0.3||uM||1.50||75.00 |- | PfuTurbo Cx||2.5||Unit/ul||1||unit||0.40||20.00 |- | 50X SYBG||50||X||0.4||X||0.40||20.00 |- | H2O||||||||||11.20||560.00 |- | Total||||||||||50.00|| |} :- Aliquot 20ul, add 30ul of bisulfite-treated adaptor-ligated DNA ::''95C for 2min --> [95C for 20sec -> '''60C for 30sec''' -> 72C for 1min] X 24 cycles --> 72C for 2min''<br> * I stopped 1st round PCR at 17 cycles as the qPCR curve was close to saturate. * I was still worried about the cycle number as 17 cycles was much less than 25 cycles by Tang's group. However, I could not let it over amplified of adaptor dimers as well. ::- I took 5ul of 1st PCR product of all samples for PAGE verification. * This might take a lot of time and TBE gel to verify. However, I want to observe the result in every single step to be able to interpret the result and get optimum condition for scRRBS. ==== 1st PCR at 17 cycles, 5ul PCR product ==== [[File:ZhangLab_2 2014-12-22 14hr 53min_1stPCR-gel1.jpg| 450px]]<br> <br> [[File:ZhangLab_2 2014-12-22 14hr 53min_1stPCR-gel2.jpg| 450px]]<br> <br> [[File:ZhangLab_2 2014-12-22 14hr 53min_1stPCR-gel3.jpg| 450px]]<br> * All samples in 3 images above (W_1 to W_16 and T_1 to T_16) had 60fg of unmeth lambda DNA spike in [[File:ZhangLab_2 2014-12-22 14hr 53min_1stPCR-gel4.jpg|350px]]<br> <br> [[File:ZhangLab_2 2014-12-22 14hr 53min_1stPCR-gel5.jpg| 320px]] * NTC_1 and NTC_2 are NTC during cell lysis step. * Idid not see any different between 1nu reaction (W_XX and T_XX) and NTC in cell lysis, MspI and ligation control. Most of the signals came from primers, adaptors and adaptor dimers and faint band below 200bp band. * However, for reaction with 10 and 100nuclei (T10n and T100n) I noticed a faint smear below and above 200bp as cropped in image. In addition, the intensity of smear was proportional to number of nuclei in the reaction. I was hoping that it is the real signal of RRBS libraries from mouse nuclei. * The next challenging for single nuclei reaction was how to remove the background of adaptor dimers ~125bp which was a dominant background and the size close to the size of expected libraries and increase amplification of the real signal which was not visible in TBE gel. * I continued to purify the 1st round amplicons with AMPure beads twice (1:1 ratio). Left over PCR product volume was 45 ul. ==== <u>AMPure bead purification</u> ==== '''''<span style="color:crimson"><u>Prep</u></span>'''''<br> - Freshly prepare 80% EtOH :- Add 45ul AMPure bead. Mix by pipetting 10x :- Sit for 8min :- Transfer to sit on magnet for 5min :- Wash twice with 200ul freshly prepared 80% EtOH :- Dry the bead for 3-5min :- Resuspend with 50 H2O :- Add 50ul of fresh AMPure bead. Mix by pipetting 10x :- Sit for 5min :- Transfer to sit on magnet for 5min :- Wash twice with 200ul freshly prepared 80% EtOH :- Dry the bead for 3-5min <u>(make sure that the beads are completely dried out to avoid EtOH inhibiting PCR)</u> :- Resuspend the bead with 40ul H2O :- Transfer purified 1st round amplicons to 8-tube strip with cap :- Sit the strip tube on magnet before adding to the 2nd round PCR to avoid bead contamination in PCR ::- 32ul of bead purified 1st round amplicons will be added to the 2nd round PCR (32/40 -> 40%) ::- I saved the rest of bead purified 1st round amplicons ==== <u>2nd round PCR</u> ==== '''''<span style="color:crimson"><u>Prep</u></span>'''''<br> * I did qPCR in small volume (10ul in duplicates) to monitor PCR cycle number. :{| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="width:80px;background:#f0f0f0;"|'''Conc''' | align="center" style="width:80px;background:#f0f0f0;"|'''unit''' | align="center" style="width:80px;background:#f0f0f0;"|'''Final conc.''' | align="center" style="width:80px;background:#f0f0f0;"|'''unit''' | align="center" style="width:80px;background:#f0f0f0;"|'''Volume (ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''50 rxn mix''' |- | Purified 1st round DNA||||||||||6.00||0.00 |- | 5X Phusion HF buffer||5||X||1||X||4.00||200.00 |- | dNTP mix||10||mM||0.3||mM||0.60||30.00 |- | TruS_F/R||10||uM||0.3||uM||0.60||30.00 |- | 50X SYBR||50||X||0.4||X||0.16||8.00 |- | Phusion HF||2||unit/ul||||||0.20||10.00 |- | H2O||||||||||8.44||422.00 |} :- Aliquot 14ul + 6ul of template ::''98C for 2min --> [98C for 10sec -> '''60C for 30sec''' -> 72C for 1min] X XX cycles --> 72C for 2min''<br> ::- I stopped 2nd PCR af different cycle number based on qPCR curve. Do not let it saturated. :- Strip 1: W_1 to W_8 :- Strip 2: W_9 to W_16 :- Strip 3: T_1 to T_8 :- Strip 4: T_9 to T_16 :- Strip 5: T10n, T100n, T0n, CTT+lamb_1, CTT+lamb_2, NTC+lamb_1, NTC+lamb_2 :- Strip 6: CTT-lamb_1, CTT-lamb_2, MspI-lamb_NTC_1, MspI-lamb_NTC_2, Lig-lamb_NTC_1, Lig-lamb_NTC_2 * I stopped reaction at different cycle number :- Strip 1-4, 6 stopped at 9 and 11 cycles :- Strip 5: stopped at 8 and 9 cycles. :- I verified 5ul of PCR product in 6% TBE gel. [[File:ZhangLab_2 2014-12-22 18hr 25min_2ndPCR-gel1.jpg| 450px]]<br> <br> [[File:ZhangLab_2 2014-12-22 18hr 25min_2ndPCR-gel2.jpg| 450px]]<br> <br> [[File:ZhangLab_2 2014-12-22 18hr 25min_2ndPCR-gel3.jpg| 450px]]<br> <br> * All samples in 3 images above (W_1 to W_16 and T_1 to T_16) had 60fg of unmeth lambda DNA spike in [[File:ZhangLab_2 2014-12-22 18hr 25min_2ndPCR-gel5.jpg| 400px]]<br> <br> [[File:ZhangLab_2 2014-12-22 18hr 25min_2ndPCR-gel6.jpg| 400px]]<br> <br> [[File:ZhangLab_2 2014-12-22 18hr 25min_2ndPCR-gel4.jpg| 380px]]<br> <br> * To confirm that the intensity of reaction with 1 nucleus was higher than NTC_1 and NTC_2 (with lambda DNA spike in), I loaded some samples of wild type (W_1 to W_5) and TKO (T_1 to T_4) at 9 cycles. I did not have NTC_1 and NTC_2 with lambda DNA spike in at 11 cycles for comparison. [[File:ZhangLab_2 2014-12-22 18hr 25min_2ndPCR-gel7_verify-2NTC-5WT-4TKO-at9cycles.jpg| 500px]]<br>
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