Chris:LabNotes/FateMapping/Calendar/2015/2015-8-11
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In-tube MDA on C1 amplicons[edit]
Background[edit]
- We will perform mass probe capture on all the single cell amplicons obtained from BA9 from the two patients we ran the DNA-seq on yesterday (WZTO BA9 and P44G BA9). We'll need to have ample material to run four different probe capture reactions similar to that done on <http://genome-tech.ucsd.edu/LabNotes/index.php/Chris:LabNotes/FateMapping/Calendar/2015/2015-7-27>:
Hemo - 9/22/2014 Probes Phusion - 9/22/2014 Probes Hemo - 5/23/2015 Probes Phusion - 5/23/2015 Probes
- Today, I'll be usng the following positive amplicons from the two C1 run on <http://genome-tech.ucsd.edu/LabNotes/index.php/Chris:LabNotes/FateMapping/Calendar/2015/2015-8-10>
- WZTO BA9
Well # C1 Rxn # Concentration Single Cell? ---------------Strip A1--------------- B1 09 13.704 Yes C1 15 13.952 Yes D1 21 13.523 Yes E1 25 13.001 Yes F1 31 16.406 Yes G1 37 13.257 Yes C2 14 11.000 Yes D2 20 13.812 Yes ---------------Strip A2--------------- E2 26 14.155 Yes C3 13 12.314 Yes E3 27 12.841 Yes F3 33 14.184 Yes D7 24 14.905 Yes E7 28 14.533 Yes G7 40 13.821 Yes H7 46 11.957 Yes ---------------Strip A3--------------- C8 17 6.037 Yes D8 23 6.790 Yes H8 47 10.075 Yes C9 16 10.221 Yes D9 22 11.334 Yes E9 30 11.339 Yes NTC (Water) NTC (Water)
- P44G BA9
Well # C1 Rxn # Concentration Single Cell? ---------------Strip B1--------------- B1 09 16.070 Yes C1 15 15.929 Yes G1 37 17.105 Yes H1 43 17.676 Yes C2 14 18.348 Yes F2 32 17.955 Yes H2 44 17.686 Yes H3 45 18.600 Yes ---------------Strip B2--------------- B4 55 19.471 Yes E4 75 17.361 Yes D5 68 20.648 Yes E5 74 19.407 Yes E6 73 16.314 Yes A7 06 14.992 Yes B7 12 15.906 Yes F7 34 16.735 Yes ---------------Strip B3--------------- A8 05 13.423 Yes E8 29 17.211 Yes A9 04 14.023 Yes H10 96 11.671 Yes C11 65 12.849 Yes B12 60 5.804 Yes F12 82 14.363 Yes H12 94 13.762 Yes
Procedure[edit]
- Make ALS and UV (also UV NS = NS is from Andrew w/ 600mM HCl):
2ul EDTA 90ul H2O+DTT (this is a premix in the ".11M DTT" tubes from Andrew) 8ul 5M KOH
- We will be doing 24 rxn's + 2 NTC:
Rxn Template 1->8, Strip A1 1ul C1 amplicon (~10ng) 1->8, Strip A2 1ul C1 amplicon (~10ng) 1->6,2NTC Strip A3 1ul C1 amplicon (~10ng) <- 2NTCs are just 1ul of water 1->8, Strip B1 1ul C1 amplicon (~10ng) 1->8, Strip B2 1ul C1 amplicon (~10ng) 1->8, Strip B3 1ul C1 amplicon (~10ng)
- Add 1.5ul ALS to each rxn and incubate on ice for 10min
- Add 1.5ul NS and proceed to adding in mastermix
- Rxn mastermix
1x 51x Template 1ul - ALS 1.5 - NS 1.5 - Mod N6 (1mM) 1 51 dNTP 0.8 40.8 10x Buffer 2 102 Phi29 1 51 Water 11.2 571.2 ---------------------------- 20 (add 16ul to each rxn)
- Aliquot 102ul per well in strip tube and add 16ul to each reaction
- MDA program (can do in regular thermocycler): 37C 2hr -> 10C forever
Target Capture - Using C1 amplicons and extended MDA from 2015_08_10 C1 run (WZTO BA9, P44G BA9) w/ 9/22/2014 probes (Hemo and Phusion Reactions)[edit]
Background[edit]
- Above, I did extended 2hr MDA on 22 amplicons from WZTO BA9 and 24 amplicons from P44G BA9
- Today, I'll be running a probe capture on all of these amplicons using my 9/22/2014 probes. Today, I'll be keeping the same probe set for each sample but using either Hemo or Phusion in roder to do the reactions
Procedure[edit]
Thermocycler Overview[edit]
1 = 95C 8:00 2 = 94.8C 0:30 -0.2C per cycle 3 = Goto 2, 173 times 4 = 60C Forever (20hr -> add KLN or Phusion -> 4hr) 5 = 94C 2:00 6 = 37C 2:00:00 7 = 95C 5:00 8 = 10C Forever
Calculation of mass of probe required for each human gDNA input[edit]
- Can find calculation on my Google Doc at <https://docs.google.com/spreadsheets/d/10hhMyThBT2nAvxIodQq_1q0RQopnfMlNzSEZEexi0nE/edit?usp=sharing>
- For each of the tests, the following is the probe and target masses required: (this will be for all samples including NTC for both Hemo and Phusion reactions)
rxn Target (ng) Probe:Target Probe (ng) Description -- 50ng 500:1 3.69 (CW 9/22) CW 9/22 Extended C1 Well -- (Hemo KlenTaq or Phusion)
- My CW 9/22/2014 probes are already 1ng/ul, so no need to dilute
- We approximated the concentration of the extended MDA to ~50ng/ul so it'll be quicker to add just 1ul of extended MDA product instead of modulating volume
rxn Target (ul) Probe (ul) Water (ul) Description 1->8 A1-3,B1-3 1.00 (Extended C1) 3.69 (CW 9/22) 13.31 CW 9/22 Extended C1 Wells (Hemo KlenTaq or Phusion)
- Final reaction volume will be 18ul for all reaction prior to adding 10x AmpLigase buffer
- We make a reaction mastermix with probes, water and AmpLigase buffer without any template and aliquot it out into a strip tube to make it quicker to pipette:
100x Target -- CW 9/22/2014 Probes 369ul Water 1331ul 10x AmpLigase Buffer 200ul
- Aliquot 235.6ul per well in a strip tube and use multi-channel pipet to pipet 19ul to each tube in a 96-well plate prior to adding 1ul of template into each
Probe+Template+Buffer Mix[edit]
- Add 2.00ul of 10x AmpLigase buffer to all rxn's
- Overlay with 40ul mineral oil (can centrifuge in order for oil and solution to separate)
- Thermocycler: 95C 8min -> cool down to 60C at 0.02C/sec -> Incubate 60C 18hr
- Note: Today, I'll be using a 96 well plate in order to do capture with the following format:
1 2 3 4 5 6 7 8 9 10 11 12 A ---------------------------------1------------------------------------ B | C V D A1 A2 A3 B1 B2 B3 A1 A2 A3 B1 B2 B3 E | F | G V H ---------------------------------8------------------------------------- ----------Hemo-------------------|-----------------Phusion-------------