Editing
Blue:RNA-Seq Experiments:totoRNAseq optimizations
Jump to navigation
Jump to search
Warning:
You are not logged in. Your IP address will be publicly visible if you make any edits. If you
log in
or
create an account
, your edits will be attributed to your username, along with other benefits.
Anti-spam check. Do
not
fill this in!
== Experimental Overview == [[File:TotoRNAseq expanded optimization3.jpg|600px]] == Purpose == *Systematic analysis of different methods for addition of forward ILMN sequencing adapter to totoRNAseq libraries *Comparison of Mg-based and RNaseIII based fragmentation methods == RNA-Seq Library Information == {| {{table}} | align="center" style="background:#f0f0f0;"|'''Library ID''' | align="center" style="background:#f0f0f0;"|'''Sample''' | align="center" style="background:#f0f0f0;"|'''Condition''' | align="center" style="background:#f0f0f0;"|'''5' Adaptor''' | align="center" style="background:#f0f0f0;"|'''3' Adaptor''' | align="center" style="background:#f0f0f0;"|'''N2 PCR Rev Primer''' | align="center" style="background:#f0f0f0;"|'''RNA-Seq Protocol''' |- | BL_totoRNAseq_1ngUHRR_||2x1ng UHRR||2min Mg Frag/Enzymatics PAP 5 min||TSO.r06||T30VN_id1||N2.id1-2||totoRNAseq |- | ||2x100pg UHRR||2min Mg Frag/Enzymatics PAP 5 min||TSO.r06||T30VN_id1||N2.id3-4||totoRNAseq |- | ||10x10pg UHRR (combined)||2min Mg Frag/Enzymatics PAP 5 min||TSO.r06||T30VN_id1-10||N2.id5||totoRNAseq |- | ||NTC||2min Mg Frag/Enzymatics PAP 5 min||TSO.r06||T30VN_id1||N2.id6||totoRNAseq |- | ||2x1ng UHRR||0.1U RNase3 Frag/Affymetrix PAP 10 min||TSO.r06||T30VN_id1||N2.id7-8||totoRNAseq |- | ||2x100pg UHRR||0.1U RNase3 Frag/Affymetrix PAP 10 min||TSO.r06||T30VN_id1||N2.id9-10||totoRNAseq |- | ||10x10pg UHRR (combined)||0.05U RNase3 Frag/Affymetrix PAP 4 min||TSO.r06||T30VN_id1-10||N2.id11||totoRNAseq |- | ||NTC||0.1U RNase3 Frag/Affymetrix PAP 10 min ||TSO.r06||T30VN_id1||N2.id12||totoRNAseq |- | ||2x1ng UHRR||2min Mg Frag/Enzymatics PAP 5 min||C1-P1-RNA-TSO||C1-P1-T31||C1-TN5-x||totoRNAseq/UMI-STRT-Seq |- | ||2x100pg UHRR||2min Mg Frag/Enzymatics PAP 5 min||C1-P1-RNA-TSO||C1-P1-T31||C1-TN5-x||totoRNAseq/UMI-STRT-Seq |- | ||3x10pg UHRR||2min Mg Frag/Enzymatics PAP 5 min||C1-P1-RNA-TSO||C1-P1-T31||C1-TN5-x|||totoRNAseq/UMI-STRT-Seq |- | ||NTC||2min Mg Frag/Enzymatics PAP 5 min||C1-P1-RNA-TSO||C1-P1-T31||C1-TN5-x||totoRNAseq/UMI-STRT-Seq |- | ||2x1ng UHRR||0.1U RNase3 Frag/Affymetrix PAP 10 min||C1-P1-RNA-TSO||C1-P1-T31||C1-TN5-x||totoRNAseq/UMI-STRT-Seq |- | ||2x100pg UHRR||0.1U RNase3 Frag/Affymetrix PAP 10 min||C1-P1-RNA-TSO||C1-P1-T31||C1-TN5-x||totoRNAseq/UMI-STRT-Seq |- | ||3x10pg UHRR||0.05U RNase3 Frag/Affymetrix PAP 5 min||C1-P1-RNA-TSO||C1-P1-T31||C1-TN5-x||totoRNAseq/UMI-STRT-Seq |- | ||NTC||0.1U RNase3 Frag/Affymetrix PAP 10 min||C1-P1-RNA-TSO||C1-P1-T31||C1-TN5-x||totoRNAseq/UMI-STRT-Seq |- | ||2x1ng UHRR||2min Mg Frag/Enzymatics PAP 5 min||Smart2_TSO||Smart2_T30VN||N7xx/N5xx||totoRNAseq/Smart-Seq2 |- | ||2x100pg UHRR||2min Mg Frag/Enzymatics PAP 5 min||Smart2_TSO||Smart2_T30VN||N7xx/N5xx||totoRNAseq/Smart-Seq2 |- | ||3x10pg UHRR||2min Mg Frag/Enzymatics PAP 5 min||Smart2_TSO||Smart2_T30VN||N7xx/N5xx||totoRNAseq/Smart-Seq2 |- | ||NTC||2min Mg Frag/Enzymatics PAP 5 min||Smart2_TSO||Smart2_T30VN||N7xx/N5xx||totoRNAseq/Smart-Seq2 |- | ||2x1ng UHRR||0.1U RNase3 Frag/Affymetrix PAP 10 min||Smart2_TSO||Smart2_T30VN||N7xx/N5xx||totoRNAseq/Smart-Seq2 |- | ||2x100pg UHRR||0.1U RNase3 Frag/Affymetrix PAP 10 min||Smart2_TSO||Smart2_T30VN||N7xx/N5xx||totoRNAseq/Smart-Seq2 |- | ||3x10pg UHRR||0.05U RNase3 Frag/Affymetrix PAP 4 min||Smart2_TSO||Smart2_T30VN||N7xx/N5xx||totoRNAseq/Smart-Seq2 |- | ||NTC||0.1U RNase3 Frag/Affymetrix PAP 10 min||Smart2_TSO||Smart2_T30VN||N7xx/N5xx||totoRNAseq/Smart-Seq2 |- | ||2x1ng UHRR||2min Mg Frag/Enzymatics PAP 5 min||Quartz_Tagging||Quartz-RT||Quartz-Suppression||totoRNAseq/Quartz-Seq |- | ||2x100pg UHRR||2min Mg Frag/Enzymatics PAP 5 min||Quartz_Tagging||Quartz-RT||Quartz-Suppression||totoRNAseq/Quartz-Seq |- | ||3x10pg UHRR||2min Mg Frag/Enzymatics PAP 5 min||Quartz_Tagging||Quartz-RT||Quartz-Suppression|||totoRNAseq/Quartz-Seq |- | ||NTC||2min Mg Frag/Enzymatics PAP 5 min||Quartz_Tagging||Quartz-RT||Quartz-Suppression||totoRNAseq/Quartz-Seq |- | ||2x1ng UHRR||0.1U RNase3 Frag/Affymetrix PAP 10 min||Quartz_Tagging||Quartz-RT||Quartz-Suppression||totoRNAseq/Quartz-Seq |- | ||2x100pg UHRR||0.1U RNase3 Frag/Affymetrix PAP 10 min||Quartz_Tagging||Quartz-RT||Quartz-Suppression||totoRNAseq/Quartz-Seq |- | ||3x10pg UHRR||0.05U RNase3 Frag/Affymetrix PAP 5 min||Quartz_Tagging||Quartz-RT||Quartz-Suppression||totoRNAseq/Quartz-Seq |- | ||NTC||0.1U RNase3 Frag/Affymetrix PAP 10 min||Quartz_Tagging||Quartz-RT||Quartz-Suppression||totoRNAseq/Quartz-Seq |- | ||2x1ng UHRR||2min Mg Frag/Enzymatics PAP 5 min||Lig_NNNX4_id01-2||MARS_T20N_id01||n/a||totoRNAseq/MARS-Seq |- | ||2x100pg UHRR||2min Mg Frag/Enzymatics PAP 5 min||Lig_NNNX4_id03-4||MARS_T20N_id01||n/a||totoRNAseq/MARS-Seq |- | ||10x10pg UHRR (combined)||2min Mg Frag/Enzymatics PAP 5 min||Lig_NNNX4_id05||MARS_T20N_id01-10||n/a|||totoRNAseq/MARS-Seq |- | ||NTC||2min Mg Frag/Enzymatics PAP 5 min||Lig_NNNX4_id06||MARS_T20N_id01||n/a||totoRNAseq/MARS-Seq |- | ||2x1ng UHRR||0.1U RNase3 Frag/Affymetrix PAP 10 min||Lig_NNNX4_id07-8||MARS_T20N_id01||n/a||totoRNAseq/MARS-Seq |- | ||2x100pg UHRR||0.1U RNase3 Frag/Affymetrix PAP 10 min||Lig_NNNX4_id9-10||MARS_T20N_id01||n/a||totoRNAseq/MARS-Seq |- | ||10x10pg UHRR (combined)||0.05U RNase3 Frag/Affymetrix PAP 5 min||Lig_NNNX4_id11||MARS_T20N_id01-10||n/a||totoRNAseq/MARS-Seq |- | ||NTC||0.1U RNase3 Frag/Affymetrix PAP 10 min||Lig_NNNX4_id12||MARS_T20N_id01||n/a||totoRNAseq/MARS-Seq |- |} Primer sequences [http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNAseq_Primer_List#totoRNA_5.27_primer_sequence] [[Blue:totoRNAseq Protocol Schemes|totoRNAseq Protocol Schemes]] == cDNA Preparation: RNAseIII-Based Fragmentation == :'''RNA''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |2ng/ul or 0.2ng/ul or 0.02ng/ul ||||||||||0.5ul |- |1:10E4 or 1:10E5 or 1:10E6 ERCC ||||||||||0.2ul |- |0.1uM T30VN||||||||||0.1ul |- |dH2O||||||||||0.2ul |- |Total||||||||||1ul |- |} :::''Incubate @ 72C 3 min'' :::''Incubate @ 37C 1 min'' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |10x RNaseIII buffer||||||||||0.2ul |- |RNase III||||||||||0.1ul |- |dH20||||||||||0.7ul |- |Total||||||||||2ul |- |} :::''Incubate @ 37C 1 min'' :'''3' Tailing''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |fRNA||||||||||2ul |- |5x RT buffer||||||||||0.5ul |- |1mM ATP||||||||||0.5ul |- |600U/ul Affymetrix PAP||||||||||0.2ul |- |40U/ul Rnase Inhibitor||||||||||0.2ul |- |dH2O||||||||||0.6ul |- |Total||||||||||4ul |- |} :::''Incubate @ 37C 4-10 min'' :::''Add 1ul of 0.1uM T30VN.id [totoRNAseq]'' :::''Add 1ul of 12uM C1-P1-T31 [UMI-STRT-Seq]'' :::''Add 1ul of 0.1uM Quartz-RT [Quartz-Seq]'' :::''Add 1ul of 2uM MARS_T20N_id [MARS-Seq]'' :::''Add 1ul of ?uM ? [Nugen Ovation]'' :'''Reverse Transcription''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |PAP RNA ||||||||||5ul |- |5xRT Buffer||||||||||1.5ul |- |10mM dNTP Mix||||||||||1ul |- |100mM DTT||||||||||0.25ul |- |Betaine (5M)||||||||||2ul |- |Smarter MMLV (200U/ul)||||||||||0.25ul |- |Total||||||||||10ul |- |} :::''Incubate @ 42C 30 min'' :::''For MARS-seq - continue to that section'' :'''Beads Purification''' ::#Add 15ul beads per well ::#Allow to bind 15 min ::#Remove supernatant and wash 2x with 1ml 80% EtOH ::#Air Dry 10min or until cracking first occurs ::#Resuspend beads in 6ul dH2O, incubate 2min, transfer to new tube; vacuspin ~8min to 3ul == cDNA Preparation: Mg-based Fragmentation == :'''RNA FRAGMENTATION''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |2ng/ul or 0.2ng/ul or 0.02ng/ul ||||||||||0.5ul |- |1:10E4 or 1:10E5 or 1:10E6 ERCC ||||||||||0.2ul |- |5x RT buffer||||||||||1ul |- |dH2O||||||||||0.3ul |- |Total||||||||||2ul |- |} :::''Incubate @ 94C 2 min'' :::''Transfer to ice'' :'''End Repair''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |fRNA||||||||||2ul |- |1mM ATP||||||||||0.4ul |- |100mM DTT||||||||||0.1ul |- |RNase Inhibitor||||||||||0.1ul |- |PNK||||||||||0.5ul |- |Total||||||||||3.1ul |- |} :::''Incubate @ 37C 30 min'' :'''3' Tailing''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |PNK_fRNA||||||||||3.1ul |- |1mM ATP||||||||||0.5ul |- |0.5U/ul Enzymatics PAP (1:10d in H2O)||||||||||0.5ul |- |Total|||||||||| 4.1 |- |} :::''Incubate @ 37C 5 min'' :::''Transfer to Ice'' :::''Add 1ul of 0.1uM T30VN.id'' :'''Reverse Transcription''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |PAP RNA ||||||||||5ul |- |5xRT Buffer||||||||||1ul |- |10mM dNTP Mix||||||||||1ul |- |100mM DTT||||||||||0.25ul |- |Betaine (5M)||||||||||2ul |- |Smarter MMLV (200U/ul)||||||||||0.25ul |- |dH2O||||||||||0.5ul |- |Total||||||||||10.1ul |- |} :::''Incubate @ 42C 30 min'' :::''For MARS-seq - continue to that section'' :'''Beads Purification''' ::#Add 15ul beads per well ::#Allow to bind 15 min ::#Remove supernatant and wash 2x with 1ml 80% EtOH ::#Air Dry 10min or until cracking first occurs ::#Resuspend beads in 6ul dH2O, incubate 2min, transfer to new tube; vacuspin ~8min to 3ul == totoRNASeq Library Preparation == :'''TSO Reaction''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |Purified cDNA||||||||||3ul |- |5x RT buffer [MMLV]||||||||||2ul |- |10mM dNTP mix||||||||||1ul |- |100mM DTT||||||||||0.25ul |- |10uM TSO||||||||||1ul |- |Betaine (5M)||||||||||2ul |- |RNase Inhibitor||||||||||0.25ul |- |Smarter MMLV||||||||||0.5ul |- |Total||||||||||10ul |- |} :::''Incubate @42C 20min'' :::''Incubate @70C 10min'' :'''PCR (PhusionHF)''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |TSO Reaction||||||||||10ul |- |2x Buffer||||||||||25ul |- |10uM P1-STRT||||||||||1ul |- |10uM PCR_R_N2_id||||||||||1ul |- |H2O||||||||||13ul |- |Total||||||||||50ul |- |} ::{| {{table}} |- |98C 30 sec|||||||||| |- |98C 15s -> 58C 20s -> 72C 30s||||||||||6x |- |98C 10s -> 72C 20s||||||||||9x (1ng); 12x (100pg) |- |72C, 5min |- |} *Run 5ul on gel: Results: == UMI-STRT-Seq Library Preparation == :'''TSO Reaction''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |Purified cDNA||||||||||3ul |- |5x First Strand buffer||||||||||1.4ul |- |10mM dNTP mix||||||||||0.7ul |- |100mM DTT||||||||||0.175ul |- |10uM C1-P1-RNA-TSO||||||||||0.7ul |- |RNase Inhibitor||||||||||0.175ul |- |SuperScriptII||||||||||0.7ul |- |dH2O||||||||||0.15ul |- |Total||||||||||7ul |- |} :::''Incubate @42C 20min'' :::''Incubate @70C 10min'' :'''PCR (Advantage)''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |TSO Reaction||||||||||7ul |- |10x Advantage 2 Buffer||||||||||7ul |- |12uM C1-P1-PCR-2||||||||||2.8ul |- |dNTPs||||||||||2.8ul |- |Advantage 2 Polymerase||||||||||2.8ul |- |H2O||||||||||47.6ul |- |Total||||||||||70ul |- |} ::{| {{table}} |- |95C 60 sec|||||||||| |- |95C 20s -> 58C 4min -> 68C 6min||||||||||5x |- |95C 20s -> 64C 30s -> 68C 1min||||||||||10x (1ng); 13x (100pg); 16x (10pg) |- |72C, 10min |- |} *Run 5ul on gel: Results: == Smart-Seq2 Library Preparation == :'''TSO Reaction''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |Purified cDNA||||||||||3ul |- |5x First Strand buffer||||||||||2ul |- |10mM dNTP mix||||||||||1ul |- |100mM DTT||||||||||0.5ul |- |100uM Smart2-TSO||||||||||0.1ul |- |MgCl2 (1M)||||||||||0.06ul |- |Betaine (5M)||||||||||2ul |- |RNase Inhibitor||||||||||0.25ul |- |SuperScriptII||||||||||0.5ul |- |dH2O||||||||||0.59ul |- |Total||||||||||10ul |- |} :::''Incubate @42C 20min'' :::''Incubate [@50C 2min > 42C 2min] x10 cycles'' :::''Incubate @70C 10min'' :::''Incubate @10C hold'' :'''PCR (KAPA-HiFi)''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |TSO Reaction||||||||||10ul |- |2x KAPA HiFi Hotstart ReadyMix||||||||||12.5ul |- |IS PCR Primer (10uM)||||||||||0.25ul |- |H2O||||||||||2.25ul |- |Total||||||||||25ul |- |} ::{| {{table}} |- |98C 3min|||||||||| |- |98C 20s -> 67C 15s -> 72C 1min||||||||||15x (1ng); 18x (100pg); 21x (10pg) |- |72C, 5min |- |} == Quartz-Seq Library Preparation == '''RT Primer Removal''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |Purified cDNA||||||||||3ul |- |10x PCR Buffer||||||||||0.4ul |- |10x Exo I buffer||||||||||0.2ul |- |100mM DTT||||||||||0.2ul |- |Exonuclease I||||||||||0.6ul |- |dH2O||||||||||1.6ul |- |Total||||||||||6ul |- |} ''Note: can add directly to beads and transfer to new tube'' :::''Incubate @37C 30min'' :::''Incubate @80C 20min'' :::''Incubate @4C'' '''Poly-A Tailing''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |ExoI RXN||||||||||6ul |- |10x PCR Buffer||||||||||0.5ul |- |100mM dATP||||||||||0.15ul |- |RNaseH||||||||||0.12ul |- |TdT enzyme||||||||||0.42ul |- |dH2O||||||||||3.81ul |- |Total||||||||||11ul |- |} :::''Incubate @37C 50 sec'' :::''Incubate @65C 10min'' :::''Incubate @4C'' '''Second Strand Synthesis''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |Poly-A Tailed Sample||||||||||11ul |- |2x Terra Direct PCR Buffer||||||||||25ul |- |10uM Tagging Primer||||||||||0.32ul |- |Terra Polymerase||||||||||2ul |- |dH2O||||||||||18.68ul |- |Total||||||||||57ul |- |} :::''Incubate @98C 2min 10 sec'' :::''Incubate @40C 1min'' :::''Incubate @68C 5min'' :::''Transfer to ice'' '''PCR Amplification''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |2nd Strand Sample||||||||||57ul |- |2x Terra Direct PCR Buffer||||||||||25ul |- |100uM PCR Primer||||||||||1ul |- |dH2O||||||||||24ul |- |Total||||||||||107ul |- |} ::{| {{table}} |- |68C 1 sec|||||||||| |- |98C 10s -> 65C 15s -> 68C 5min||||||||||15x (1ng); 18x (100pg); 21x (10pg) |- |68C, 5min |- |} == MARS-Seq Library Preparation == '''Exonuclease Digestion''' :::''Add 1ul Exonuclease I (1:10 diluted in 1x exonuclease buffer)to each RT reaction'' :::''Incubate at 37C for 30min'' :::''Incubate at 80C for 10min'' :''Beads Purification:'' ::#Add 15ul beads per well ::#Allow to bind 15 min ::#Remove supernatant and wash 2x with 1ml 80% EtOH ::#Air Dry 10min or until cracking first occurs ::#Resuspend beads in 17ul 10mM Tris-HCl pH7.5, incubate 2min, transfer to new tube '''Second Strand Synthesis''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |cDNA||||||||||17ul |- |10x NEB second strand synthesis buffer||||||||||2ul |- |Second strand synthesis enzyme||||||||||1ul |- |Total||||||||||20ul |- |} :::''Incubate @ 16C for 2.5 hours'' '''IVT''' ::#Add 28ul beads per well ::#Allow to bind 15 min ::#Remove supernatant and wash 2x with 1ml 80% EtOH ::#Air Dry 10min or until cracking first occurs ::#Resuspend beads in: ::{| {{table}} |- |||||||||||<u>Volume</u> |- |10x Reaction Buffer (T7 high yield NEB)||||||||||2ul |- |UTP||||||||||2ul |- |ATP||||||||||2ul |- |CTP||||||||||2ul |- |GTP||||||||||2ul |- |T7 polymerase||||||||||2ul |- |dH2O||||||||||8ul |- |Total||||||||||20ul |- |} :::''Incubate 16 hours (overnight) @ 37C''' :::''Add 1ul Turbo DNase I and incubate @ 37C 15min :''Beads Purification:'' ::#Add 24ul beads per well ::#Allow to bind 15 min ::#Remove supernatant and wash 2x with 1ml 80% EtOH ::#Air Dry 10min or until cracking first occurs ::#Resuspend beads in 10ul dH20, transfer to new tube, vacuspin to 3ul '''Adaptor Ligation''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |aRNA||||||||||3ul |- |100uM Lig_NNNX4_idx||||||||||1ul |- |} :::''Incubate @ 70C for 3min'' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |aRNA/adaptor||||||||||4ul |- |10x T4 Ligase buffer||||||||||1ul |- |DMSO||||||||||0.95ul |- |10mM ATP||||||||||1ul |- |PEG8000||||||||||2ul |- |T4 RNA Ligase I(10U/ul)||||||||||1ul |- |dH2O||||||||||0ul |- |Total||||||||||10ul |- |} :::''Incubate at 22C for 2hrs'' '''Reverse Transcription''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |Ligated RNA||||||||||8ul |- |10uM MARS_RT_2||||||||||1ul |- |10mM dNTP||||||||||1ul |- |Total||||||||||10ul |- |} :::''Incubate @ 65C 5min'' :::''Incubate on ice 1min'' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |RNA/Primer||||||||||10ul |- |10x RT Buffer||||||||||2ul |- |25mM MgCl2||||||||||0.8ul |- |100mM DTT||||||||||2ul |- |RNase Inhibitor (40U/ul)||||||||||1ul |- |SSIII (200U/ul)||||||||||1ul |- |dH2O||||||||||3.2ul |- |Total||||||||||10ul |- |} :::''Incubate @42C 10min'' :::''Incubate @50C 50min'' :::''Incubate @85C 5min'' '''Beads Purify''' ::#Add 30ul beads per well ::#Allow to bind 15 min ::#Remove supernatant and wash 2x with 1ml 80% EtOH ::#Air Dry 10min or until cracking first occurs ::#Resuspend beads in 10ul dH20, transfer to new tube '''PCR Amplification''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |cDNA||||||||||10ul |- |SybrGreen KAPA 2x MM||||||||||25ul |- |10uM P5_Rd1_PCR_F||||||||||2.5ul |- |10uM P7_Rd2_PCR_R||||||||||2.5ul |- |dH2O|||||||||| |- |} ::{| {{table}} |- |95C 30 sec|||||||||| |- |95C 15s -> 60C 20s -> 72C 30s||||||||||12x |- |72C, 10min |- |} == Nextera XT for Quartz-Seq ==
Summary:
Please note that all contributions to ZhangLabWiki may be edited, altered, or removed by other contributors. If you do not want your writing to be edited mercilessly, then do not submit it here.
You are also promising us that you wrote this yourself, or copied it from a public domain or similar free resource (see
ZhangLabWiki:Copyrights
for details).
Do not submit copyrighted work without permission!
Cancel
Editing help
(opens in new window)
Template used on this page:
Template:Table
(
edit
)
Navigation menu
Personal tools
Not logged in
Talk
Contributions
Create account
Log in
Namespaces
Page
Discussion
English
Views
Read
Edit
View history
More
Search
Navigation
Main Page
Current events
Recent changes
Random page
Investigators
Matt Cai
Song Chen
Eric Chu
Dinh Diep
Elizabeth Duong
Shicheng Guo
Alan Fung
Daniel Jacobsen
Blue Lake
Huy Lam
Alice Li
Andrew Richards
Brandon Sos
Chris Wei
Yan Wu
Kun Zhang
Tools
What links here
Related changes
Special pages
Page information