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== Amplification == * '''TEST''' if amplification works (in small volume 25ul add 1.25ul of adapter ligated template) {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''1x rxn''' | align="center" style="background:#f0f0f0;"|'''6x rxn mix''' |- | adapter ligated DNA||1.25||0.00 |- | PCR_F(10uM)||0.50||3.00 |- | PCR_R (10uM)||0.50||3.00 |- | SYBR Green 50x||0.10||0.60 |- | H2O||10.15||60.90 |- | Phusion HF, 2xMM||12.50||75.00 |- | Total volume||25.00||150.00 |} * Aliquot 23.75ul, add 1.25 of adapter ligated DNA '''Program''' 98C - 30s, (98C - 10s, 65C - 20s, 72C - 30s)x12, 72C - 2min, hold at 15C [[File:qPCR_20130115_TESTpcr.png.png| 500px]] * PAGE analysis to see the pattern of amplified amplicons (loaded PCR product 2ul, no bead purification) [[File:ZhangLab_2 2013-01-15 21hr 12min_Test_PCR.jpg| 300px]] * Since PCR worked well, I amplified all adapter ligated DNA (B1-E1, 3 replicates, 5ul of template in 100ul reaction). I reduced number of cycle from 12 to 11 cycles {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''1x rxn''' | align="center" style="background:#f0f0f0;"|'''13.5rxn mix''' |- | adapter ligated DNA||5.00||0.00 |- | PCR_F(10uM)||2.00||27.00 |- | PCR_R.N2IndX(10uM)[X=1-4]||2.00||0.00 |- | SYBR Green 50x||0.80||10.80 |- | H2O||40.20||542.70 |- | Phusion HF, 2xMM||50.00||675.00 |- | Total volume||100.00||1350.00 |} * Aliquot 93ul, add 5 of adapter ligated DNA and 2ul of PCR_R.N2Indx '''Program''' 98C - 30s, (98C - 10s, 65C - 20s, 72C - 30s)x11, 72C - 2min, hold at 15C [[File:qPCR_20130115_3-replictes_B1-E1.png.png| 500px]] * Note: I used different types of plates (TEST PCR used the real time PCR tube (Eppendort WW), the 3 replicates used the Eppendorf plate for epMotion by set up the back ground as the clear plate) {| {{table}} | align="center" style="background:#f0f0f0;"|'''Position in<br>AC-718-384-well''' | align="center" style="background:#f0f0f0;"|'''Position in<br> AC-718-96-well_plate1''' | align="center" style="background:#f0f0f0;"|'''Individual library ID''' | align="center" style="background:#f0f0f0;"|'''Indx''' |- | J03||B1||718_plate1_B1||PCR_R.N2Indx1 |- | L02||C1||718_plate1_C1||PCR_R.N2Indx2 |- | M05||D1||718_plate1_D1||PCR_R.N2Indx3 |- | N03||E1||718_plate1_E1||PCR_R.N2Indx4 |} * Pooled 80ul of amplified amplicons from the three replicates (total 240ul) and bead purified (diluted Ampure beads 3X with 20%PEG) with epMotion. * Eluted with EB buffer total volme 75ul * Dr. Zhang suggested to pooled 50% of each library and perform PAGE size-selection (may not equal molar ratio). [[File:ZhangLab_2 2013-01-17 13hr 37min_718_plate1_B1-E1-PAGE-SS.jpg| 500px]] * I cut the band at the position between 400-600bp, resuspend sequencing library with H2O total volume 30ul. * Alan will do qRCR to quantify the library concentration * Verify the library by PAGE analysis [[File:ZhangLab_2 2013-01-17 16hr 07min_SkinMB_718_p1_N2_B1-E1_Jan15-e.jpg| 300px]] * '''Library ID: JG-SkinMB_718_p1_N2_B1-E1_Jan15.2013''' * I performed PAGE quantification on individual library (B1-E1) in the range 400-600bp for further experiment (DSN) {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Position in <br>AC-718-384-well''' | align="center" style="background:#f0f0f0;"|'''Position in <br>AC-718-96-well_plate1''' | align="center" style="background:#f0f0f0;"|'''Individual library ID''' | align="center" style="background:#f0f0f0;"|'''Conc. (ng/ul)''' | align="center" style="background:#f0f0f0;"|'''Yields in 35ul (ng)''' |- | J03||B1||718_plate1_B1||0.88||30.86 |- | L02||C1||718_plate1_C1||2.59||90.55 |- | M05||D1||718_plate1_D1||3.12||109.08 |- | N03||E1||718_plate1_E1||2.74||95.82 |} * Continued on: http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2013-1-31
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