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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/2013-3-12 == Amplification TEST == * There are two set of primers * '''Set 1''': 1 forward primer without barcode && 3 different reverse primers with different barcodes (Indx1-3) {| {{table}} | 1-1. gDNA (12878)||Indx1 |- | 2-1. Total RNA||Indx2 |- | 3-1. ds cDNA no RNasA treatment||Indx3 |- | 4-1. ds cDNA RNaseA treatment||Indx3 |} * '''Set 2''': 3 different forward primers with different barcodes && combination of 9 reverse primer without barcodes {| {{table}} | 1-2. gDNA (12878)||Indx1 |- | 2-2. Total RNA||Indx2 |- | 3-2. ds cDNA no RNasA treatment||Indx3 |- | 4-2. ds cDNA RNaseA treatment||Indx3 |} * Note: Since we have only 3 different indexes, and the two set of indexes are the same, the libraries amplified by the two sets of primers can not be pooled together and sample 3-1 and 3-2 will not combinded in the pooled === PCR TEST set up === === Set 1: by Matt === {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Sample set 1''' | align="center" style="background:#f0f0f0;"|'''Index''' | align="center" style="background:#f0f0f0;"|'''Primer names xxxxxxxxxxxxxxxxx''' |- | 1-1. gDNA (12878)||Indx1|| |- | 2-1. Total RNA||Indx2|| |- | 3-1. ds cDNA no RNasA treatment||Indx3|| |- | 4-1. ds cDNA RNaseA treatment||Indx3|| |} '''PCR Mix'''<br> {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''1 rxn''' | align="center" style="background:#f0f0f0;"|'''5.5 rxn mix''' |- | Captured template||1.00||0.00 |- | 10uM Forward no barcodes||0.40||2.20 |- | 10uM Reverse +Indx||0.40||0.00 |- | 2x KAPA SYBG fast MM||12.50||68.75 |- | H2O||10.70||58.85 |- | Total||25.00||137.50 |} * Aliqout 23.6ul, add 0.4ul of reverese primer, add 1ul of circular DNA === Set 2 : by Noi === {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Sample set 2''' | align="center" style="background:#f0f0f0;"|'''Index''' | align="center" style="background:#f0f0f0;"|'''Primer names xxxxxxxxxxxxxxxxx''' |- | 1-2. gDNA (12878)||Indx1|| |- | 2-2. Total RNA||Indx2|| |- | 3-2. ds cDNA no RNasA treatment||Indx3|| |- | 4-2. ds cDNA RNaseA treatment||Indx3|| |} '''PCR Mix'''<br> {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''1 rxn''' | align="center" style="background:#f0f0f0;"|'''5.5 rxn mix''' |- | Captured template||1.00||0.00 |- | 10uM Forward + Indx||0.40||0.00 |- | 10uM Reverse no barcodes||0.40||2.20 |- | 2x KAPA SYBG fast MM||12.50||68.75 |- | H2O||10.70||58.85 |- | Total||25.00||137.50 |} * '''Program''' 98C 30s -> (98C 10s -> 52C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x15 -> 72C 3min<br> === Set 1 === {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''1rxn''' | align="center" style="background:#f0f0f0;"|'''6.5 rxn''' |- | Captured template||12.00||0.00 |- | 10uM Forward no barcodes||2.00||13.00 |- | 10uM Reverse +Indx||2.00||0.00 |- | 2x KAPA SYBG fast MM||50.00||325.00 |- | H2O||34.00||221.00 |- | Total||100.00||650.00 |} * Aliquot 86ul, add 2ul of reverse Ind, 12ul of circular DNA * Will add more for second trial by Matt for set 2 experiment (use 10uM and 100uM of reverse primers === Set 2 === {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''''' |- | Captured template||1rxn||6.5 rxn |- | 10uM Forward + Indx||12.00||0.00 |- | 100uM Reverse no barcodes||2.00||0.00 |- | 2x KAPA SYBG fast MM||2.00||13.00 |- | H2O||50.00||325.00 |- | Total||34.00||221.00 |- | ||100.00||650.00 |} * Aliquot 86ul, add 2ul of Forward Ind, 12ul of circular DNA * '''Program''' 98C 30s -> (98C 10s -> 52C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x13 -> 72C 3min (total 21 cycles) <br> * Perform bead purification , set 1 elute total volume 60ul, set 2 elute total volume 50ul. * Verify by PAGE an do PAGE quantification to see the ratio of each product before pooling the libraries. * Note: I quantified based on the expected band ~250bp and ~400 (two copy of target based on amplification of circular DNA. [[File:ZhangLab_2 2013-03-15 11hr 27min_SeqLib_Insitu.jpg| 400px]] {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Samples''' | align="center" style="background:#f0f0f0;"|'''Conc. (ng/ul)''' | align="center" style="background:#f0f0f0;"|'''Yields(ng)''' | align="center" style="background:#f0f0f0;"|'''Pooled volume(ul)''' |- | gDNA-set1||26.50||1590.05||37.73 |- | dsDNA -RNaseA set1||34.14||2048.43||29.28 |- | dsDNA +RNaseA set1||33.33||1999.55||30.00 |- | gDNA-set2||27.11||1355.44||10.41 |- | dsDNA -RNaseA set2||7.05||352.73||40.00 |- | dsDNA +RNaseA set2||10.38||519.25||27.17 |}
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