Chris:LabNotes/FateMapping/Calendar/2014/2014-9-9
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Target Capture - Troubleshoot v6 probes (Part II)[edit]
Background[edit]
- This is continuation of <http://genome-tech.ucsd.edu/LabNotes/index.php/Chris:LabNotes/FateMapping/Calendar/2014/2014-9-8>
- As a reminder, the conditions we want to test are:
1) v6 probes, all-in-one KLN mastermix with old Epicentre dNTP rxn's 1,5 2) v6 probes, all-in-one KLN mastermix with new NEB dNTP rxn's 2,6 3) v6 probes, separate KLN mastermix with old Epicentre dNTP rxn's 3,7 4) v6 probes, separate KLN mastermix with new NEB dNTP rxn's 4,8
Procedure[edit]
- Prep two different dNTP dilutions:
- Epicentre MDA dNTP = 1/25d
- NEB dNTP = 1/10d
Rxn's 1,5 (all-in-one KLN, old Epicentre dNTP), 2ul added[edit]
Component Stock conc Final conc 1x(ul) MM Vol (5x) (ul) Hemo KlenTaq 100% 20% 0.4 2.0 AmpLigase 5U/ul 0.5U/ul 0.2 1.0 dNTP 1000uM 100uM 0.2 1.0 (MDA dNTP = 25mM, so do 1/25d) 10x AmpLigase Buff 10x 1x 0.2 1.0 H2O 1.0 5
Rxn's 2,6 (all-in-one KLN, new NEB dNTP), 2ul added[edit]
Component Stock conc Final conc 1x(ul) MM Vol (5x) (ul) Hemo KlenTaq 100% 20% 0.4 2.0 AmpLigase 5U/ul 0.5U/ul 0.2 1.0 dNTP 1000uM 100uM 0.2 1.0 (NEB dNTP = 10mM, so do 1/10d) 10x AmpLigase Buff 10x 1x 0.2 1.0 H2O 1.0 5
Rxn's 3,7 (separate KLN, old Epicentre dNTP), 4ul added[edit]
Component Stock conc Final conc 1x(ul) MM Vol (5x) (ul) Hemo KlenTaq 100% 10% 0.4 2.0 dNTP 1000uM 50uM 0.2 1.0 (MDA dNTP = 25mM, so do 1/25d) 10x AmpLigase Buff 10x 1x 0.4 2.0 H2O 2.8 14.0 AmpLigase 5U/ul 0.25U/ul 0.2 1.0
Rxn's 4,8 (separate KLN, new NEB dNTP), 4ul added[edit]
Component Stock conc Final conc 1x(ul) MM Vol (3x) (ul) Hemo KlenTaq 100% 10% 0.4 2.0 dNTP 1000uM 50uM 0.2 1.0 (NEB dNTP = 10mM, so do 1/10d) 10x AmpLigase Buff 10x 1x 0.4 2.0 H2O 2.8 14.0 AmpLigase 5U/ul 0.25U/ul 0.2 1.0
- Thermocycler (continued): Add 2ul or 4ul KLN to each rxn -> Incubate 60C 4.5hr -> 94C 2min -> add 2ul ExoI/ExoIII mix (1:1) to each rxn -> 37C 2hr -> 94C 5min -> Hold 10C
- Note: Right before 4hr is up, prep the ExoI/ExoIII mix at 1:1 ratio
- Note: Made sure to equilibrate to 37C for at least 1min before adding in Exo
Phusion PCR[edit]
- We are going to use the previous target captured circularlized DNA as template for Phusion reaction
- The mastermix used is below (based on the protocol published online File:Tech-manual-f-548-phusion-flash-high-fidelity-pcr-master-mix.pdf)
Component 1x(ul) 11x(ul) Init Conc Final Conc H2O 2.6 33 -- -- 2x Phusion Flash PCR MM 10 110 2x 1x AmpF6.4Sol 1 11 10uM .5uM AmpR6.3.Indx (Indx40) 1 11 10uM .5uM Circularized DNA Template 5 -- -- -- 20
- Also will include 2 PCR NTC's (two reactions in the third strip)
- The two rxn's are PCR NTC (just add 5ul water)
- Real-time PCR Profile: (need to change to 98C denaturing)
98C 30sec (98C 10sec -> 58C 30sec -> 72C 20sec)x8 (98C 10sec -> 72C 20sec)x10-15 cycles 72C 3min Hold 15C
Results[edit]
- Below is the gel image for this experiment
- The gel lanes are as follows:
x L 1->10 (where 9,10 are PCR NTC)
File:Cw ZhangLab 2 2014-09-09 17hr 53min positive control troubleshoot.jpg
- Looks like the positive control worked for all conditions. Perhaps it was just contamination by accidentally adding in my probes instead of v6 probes? This time around, I did do repeat pipetting whenever I added in the new mixes, so I'll make sure to continue doing that for next time.
- For now, I'll try either repeating the AmpLigase/Hemo KlenTaq in separate steps experiment again or try with the new Taq fragment that Dinh gave me or with Phusion instead of Hemo KlenTaq. However, first, I'll regroup and look into the mappability/alignability of capture arms as Dr. Zhang suggested.