Chris:LabNotes/FateMapping/Calendar/2015/2015-8-12
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Target Capture - Using C1 amplicons and extended MDA from 2015_08_10 C1 run (WZTO BA9, R55E BA9) w/ 9/22/2014 probes (Hemo and Phusion Reactions) (Part II)
Background
- This is a continuation of the experiment started on <http://genome-tech.ucsd.edu/LabNotes/index.php/Chris:LabNotes/FateMapping/Calendar/2015/2015-8-11>
- As a reminder, the reactions of rthis experiment are described below (46 C1 amplicons in total + 2NTC, all with 9/22/14 probes; one set with Hemo and another with Phusion)
Procedure
Prep KLN mix solution (6 strips, 8 rxn's each)
- We are using HemloKlenTaq for the reaction (because AmpliTaq Stoffel was discontinued)
- First, perform the following dilutions:
- Dilute dNTP 1/10d (6ul dNTP + 54ul H2O) (10mM/nt -> 1mM/nt)
- Mastermix recipe
Component Stock conc Final conc 1x(ul) MM Vol (54x) (ul) Hemo KlenTaq 100% 20% 0.4 21.6 AmpLigase 5U/ul 0.5U/ul 0.2 10.8 dNTP 1mM 100uM 0.2 10.8 10x AmpLigase Buff 10x 1x 0.2 10.8 H2O 1.0 54
- Aliquot 13ul KLN mix to each well of a strip tube so can use multichannel when adding to the reactions
- Add 2ul KLN mix to all strips using multichannel
Prep Phusion mix solution (6 strips, 8 rxn's each + 2 NTC)
- First, perform the following dilutions:
- Dilute dNTP 1/10d (6ul dNTP + 54ul H2O) (10mM/nt -> 1mM/nt) (same as above)
- Dilute NAD+ 1/10d (45ul NAD+ + 405ul H2O)(50mM -> 5mM)
- Mastermix recipe
Component Stock conc Final Amount 1x(ul) MM Vol (53x) (ul) NAD+ 5mM 40nmol 8.0 (after dil) 424 dNTP 1mM 600pmol 0.6 (after dil) 31.8 Betaine 5M 15umol 3.0 159 10x AmpLigase Buff 10x 1x 2.0 106 AmpLigase 5U/ul 10U 2.0 106 Phusion HF DNA Pol 2000U/ml 6.4U 3.2 169.6 <- We were short 40ul so had to add 40ul of water more in order to make up for the volume difference Water 1.2 63.6
- Aliquot 130 Phusion mix to each well of a strip tube so can use multichannel when adding to the reactions
- Add 20ul Phusion mix to all strips using multichannel
- Thermocycler (continued): Add KLN or Phusion mix to each rxn -> Incubate 60C 4hr -> 94C 2min
- Note: I took out tubes immediately after 94C incubation was done and put on ice for minute then add Exo mix and vortex/centrifuge then put back in thermocycler for 37C 2hr incubation
ExoI/ExoIII treatment
- We want to then add 20U ExoI and 100U ExoIII
- Mastermix recipe
Component Stock Conc Final Amount 1x(ul) MM Vol (108x) (ul) ExoI 20U/ul 20U 1 108 ExoIII 200U/ul 100U 0.5 54 Water 0.5 54
- Aliquot 26ul Exo mix to each well of a strip tube so can use multichannel when adding to the reactions
- Thermocycler (continued): Add 2ul ExoI/ExoIII mix -> 37C 2hr -> 95C 5min
Phusion PCR
- We are going to use the target captured circularized DNA as template for Phusion reaction
- Note: Since we are not going to do qPCR, we don't need to add SYBR into rxn mix
- Mastermix recipe (we'll need to decrease the PCR volume because of the higher number of reactions required and limited reagents we have on-hand; to make things simple, we'll use the same input template concentration into each PCR reaction)
Component 1x(ul) 26x(ul) Init Conc Final Conc Make two of these in order for total of 52 rxns H2O 14 364 -- -- 2x Phusion Flash PCR MM 25 650 2x 1x AmpF6.4Sol 2.5 65 10uM .5uM AmpR6.3.Indx 2.5 (add separately) 10uM .5uM Circularized DNA Template 6 (add separately) -- -- 50
- Below are the indices used for the reactions: (this is based on the spreadsheet <https://docs.google.com/spreadsheets/d/1F2L2NpqYCXYwYPk1WQdJDkj3yZKMJftYfeYUHtiXCL4/pubhtml#>)
- Note: We are overlaping indices for single cells across the two different probe sets (when combining the different libraries, make sure combine in equal amounts based on gel quantification). However, we want to make sure that the indices for the Hemo/Phusion probe capture reactions are different in order to validate that there is a difference between the two enzyme captures
---------------------------9/22/2014 Probes (Strip A)------------------------------------ rxn Index Indx Seq (RC) 1A 1 ATCACG 2A 2 CGATGT 3A 4 TGACCA <- Indx 3 is bad 4A 5 ACAGTG 5A 6 GCCAAT 6A 7 CAGATC 7A 8 ACTTGA 8A 10 TAGCTT <- Index 9 is bad ---------------------------9/22/2014 Probes (Strip B)------------------------------------ rxn Index Indx Seq (RC) 1B 11 GGCTAC 2B 12 CTTGTA 3B 13 CCATGA 4B 14 AAGACA 5B 15 CTTCCT 6B 16 GGGGTT 7B 17 CTCATC 8B 18 AGTTCA ---------------------------9/22/2014 Probes (Strip C)------------------------------------ rxn Index Indx Seq (RC) 1C 19 GACGCA 2C 20 CCTGTC 3C 21 CAACCC 4C 22 CTCGGA 5C 23 TCGAAA 6C 24 ATTCGC 7C 25 TACTGC 8C 26 TCGTGA NTC 3 TTAGGC <- Indx 3 is bad, so can use for NTC
---------------------------5/23/2015 Probes (Strip A)------------------------------------ rxn Index Indx Seq (RC) 1A 1 ATCACG 2A 2 CGATGT 3A 4 TGACCA <- Indx 3 is bad 4A 5 ACAGTG 5A 6 GCCAAT 6A 7 CAGATC 7A 8 ACTTGA 8A 10 TAGCTT <- Index 9 is bad ---------------------------5/23/2015 Probes (Strip B)------------------------------------ rxn Index Indx Seq (RC) 1B 11 GGCTAC 2B 12 CTTGTA 3B 13 CCATGA 4B 14 AAGACA 5B 15 CTTCCT 6B 16 GGGGTT 7B 17 CTCATC 8B 18 AGTTCA ---------------------------5/23/2015 Probes (Strip C)------------------------------------ rxn Index Indx Seq (RC) 1C 19 GACGCA 2C 20 CCTGTC 3C 21 CAACCC 4C 22 CTCGGA 5C 23 TCGAAA 6C 24 ATTCGC 7C 25 TACTGC 8C 26 TCGTGA NTC 3 TTAGGC <- Indx 3 is bad, so can use for NTC
- Thermocycler Profile: (need to change to 98C denaturing)
98C 30sec (98C 10sec -> 58C 30sec -> 72C 20sec)x8 (98C 10sec -> 72C 20sec)x12 cycles 72C 3min Hold 15C