Chris:LabNotes/FateMapping/Calendar/2015/2015-8-11

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In-tube MDA on C1 amplicons[edit]

Background[edit]

Hemo - 9/22/2014 Probes
Phusion - 9/22/2014 Probes
Hemo - 5/23/2015 Probes
Phusion - 5/23/2015 Probes
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]

    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-------------