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* [[http://genome-tech.ucsd.edu/LabNotes/index.php/noi:DMR220k_LabNotes '''Link to calendar''']] * Continued from: http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2013-4-24 * Received 12 positive control samples using the CGI protocol from Jeff (check details of his experiment in his page) * Note from Jeff: I amplified e. coli gDNA using standard in tube MDA, with the addition of diluted dUTP at a very low concentration. I then purified, and diluted the amplicons to 1 ng and 10 ng. I then used USER/Uracil DNA glycosylase (UDG) to remove the dUTPs incorporated during MDA, and used Exo - klenow for nick translation and A tailing. '''Sample list (4ul each)''' 2 10 ng, high USER conc. 2 10 ng, low USER conc. 2 1 ng, high USER conc. 2 1 ng, low USER conc. 2 1ng, UDG 2 10ng, UDG * To process these samples ** Do adapter ligation, bead purification, and PCR ** Require at least 10X dilution of original volume (4ul --> need minimum ligation reaction 40ul) ** If the reaction works, we expected to see a smear from ~500 -1500bp ** Since the length of a smear is large, I would increase extension time during amplification to be longer (1min) ** Since DNA input is in the range of 1-10ng, I will used the same condition I tried on 2013_04_23 but using 50ul ligation reaction '''Order of the tubes (need to check more info with Jeff)''' 1. 1ng high/low? USER short incubation 2. 1ng high/low? USER short incubation 3. 10ng high/low? USER short incubation 4. 10ng high/low? USER short incubation 5. 1ng high/low? USER long incubation 6. 1ng high/low? USER long incubation 7. 10ng high/low? USER long incubation 8. 10ng high/low? USER long incubation 9. 1ng UDG 10. 1ng UDG 11. 10ng UDG 12. 10ng UDG 13. NTC == Adapter ligation == * Adapter ligation mix (Total 50ul) : Ligation reaction mix 49.2ul/reaction & 0.8ul of 3uM adapters (Diluted 30uM adapters 10X with cold 1X Stoffel buffer) === Adapter preparation === * Prepare 30uM adapters {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''10ul, 30uM''' |- | 10XStoffel buffer||1.00 |- | 100uM PE_t_adapter||3.00 |- | 100uM PE_b_adapter||3.00 |- | H2O||3.00 |} * 94C 2min -> 0.2C/sec to 20C -> 4C hold * Dilute 10X by mixing 3ul of 30uM adapters with 27ul 1X Stoffel buffer (keep annealed adapters and buffer on cold box all the time) * Store 30uM annealed adapter at -20C for future use === Ligation reaction mix === {| class="wikitable" style="text-align:center; | align="center" style="background:#f0f0f0;"|'''Ligation reaction mix''' | align="center" style="background:#f0f0f0;"|'''1x rxn''' | align="center" style="background:#f0f0f0;"|'''13x rxn mix''' |- | KAPA 5X Ligation buffer||10.00||130.00 |- | KAPA DNA ligase||5.00||65.00 |- | H2O||20.20||262.60 |- | Total||35.20||457.60 |} * Aliquot 35.2 ul of ligation reaction mix to each tube (I prefer using a clear tube since I can see from the side during bead purification) * Transfer 4ul of sample to reaction tubes ** If the sample is already in a clear tube, just add ligation reaction mix directly to fragmented DNA and adjust volume in ligation reaction mix ** I used 0.2 ml PCR Tube Strips, low-Profile tube (Biorad) to set up reaction * Rinse the tube with 10ul H2O and transfer all to reaction tube (total volume now is 49.2ul) * Note: for NTC, I accidentally added too much H2O, so total of reaction of NTC was 60.8ul * Add 0.8ul of 3uM adapters * 20C for 15min (no shaking) * Bead purification: 50ul AmPure beads mix by pipetting 10 times, wait 10min (no shaking), sit on magnet 5min, wash twice with fresh 80% EtOH, dry for 3 min ** I was with 160ul of 80% EtOH since I used low profile tube. * Eluted with 15ul H2O (used all for PCR) == Amplification == {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''1x rxn''' | align="center" style="background:#f0f0f0;"|'''13.5 rxn mix''' |- | adapter ligated DNA||15.00||0.00 |- | PCR_Fs(10uM)||1.00||13.50 |- | PCR_R.N2IndX(10uM)||1.00||0.00 |- | H2O||8.00||108.00 |- | 2X KAPA SYBR MM||25.00||337.50 |- | Total volume||50.00||675.00 |} * Aliquot 34ul, add 1ul of PCR_R.N2Indx, 15ul of adapter ligated DNA {| {{table}} | align="center" style="background:#f0f0f0;"|'''Samples''' | align="center" style="background:#f0f0f0;"|'''Indx''' |- | 1. 1ng high/low? USER short incubation||PCR_R.N2Indx1 |- | 2. 1ng high/low? USER short incubation||PCR_R.N2Indx2 |- | 3. 10ng high/low? USER short incubation||PCR_R.N2Indx3 |- | 4. 10ng high/low? USER short incubation||PCR_R.N2Indx4 |- | 5. 1ng high/low? USER long incubation||PCR_R.N2Indx5 |- | 6. 1ng high/low? USER long incubation||PCR_R.N2Indx6 |- | 7. 10ng high/low? USER long incubation||PCR_R.N2Indx7 |- | 8. 10ng high/low? USER long incubation||PCR_R.N2Indx8 |- | 9. 1ng UDG||PCR_R.N2Indx21 |- | 10. 1ng UDG||PCR_R.N2Indx22 |- | 11. 10ng UDG||PCR_R.N2Indx23 |- | 12. 10ng UDG||PCR_R.N2Indx24 |- | 12. NTC||PCR_R.N2Indx24 |} '''Program (Eppendorf Realplex)''' 98C - 30s, (98C - 10s, 62C - 30s, 72C - 60s)x15, 72C - 2min, hold at 15C * Jeff suggested not go over 15 cycles. I stopped reaction for 10ng UDG at 13 cycles and the rest at 15 cycles to make sure that I would see amplification. '''qPCR curve''' [[File:20130425_lowinput_CGI.png| 650px]] * The qPCR curve of NTC was lower than other samples. This was not as usual since I expected to see high background of adapters especially in NTC. * PAGE analysis to verify the PCR products by loading 5 ul of PCR product without bead purification (1/10 of total volume) [[File:ZhangLab_2 2013-04-25 18hr 24min_lowinput_CGI_e.jpg| 520px]] [[File:ZhangLab_2 2013-04-25 18hr 16min_lowinput_CGI_e.jpg| 330px]] S and L: with USER UDG = Uracil DNA glycosylase * Will add more info * '''Comments from Dr. Zhang''' ** He suggested to use commercial adapter from NEB or HPLC purified adapter to avoid the effect of self ligated adapter due to the quality of adapter we used from IDT (desalted oligos). ** He suggested Jeff to increase the ratio od dUTP during MDA to minimize the size of library and tring USER with PNK to generate 3'-OH. ** Will be more details * Continued on: http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2013-4-30
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