Kun:LabNotes/MONOD/2014-11-23: Difference between revisions

From ZhangLabWiki
Jump to navigation Jump to search
Line 82: Line 82:
     95C 5min -> (95C 15sec -> 58C 30sec -> 72C 15 sec) x 30
     95C 5min -> (95C 15sec -> 58C 30sec -> 72C 15 sec) x 30
     Monitor the amplification curves and terminate the reactions before plateauing.
     Monitor the amplification curves and terminate the reactions before plateauing.
   [[Image:Selector_2014-11-25_50C.png|400px]][[Image:Selector_2014-11-25_53C.png|400px]]
 
   [[Image:Selector_2014-11-25_57C.png|400px]][[Image:Selector_2014-11-25_60C.png|400px]]
   [[Image:Selector_2014-11-25_50C.png|300px]][[Image:Selector_2014-11-25_53C.png|290px]]
 
   [[Image:Selector_2014-11-25_57C.png|290px]][[Image:Selector_2014-11-25_60C.png|300px]]

Revision as of 23:02, 26 November 2014

Selector v2 Round 2

Step 1. RE digestion

  • Materials:
    • gDNA: HAPMAP gDNA from Coriell GM18506 (diluted to 50ng/ul)
    • BfaI: NEB, R0568S, 10U/ul
    • DpnII: NEB, R0147S, 50U/ul
    • MspI: NEB, R0106S, 20U/ul
  • To save time for the future test, I would set up more reactions than I need for this first test.
                                    x 4
      gDNA (50ng/ul)           8 ul
      10x CutSmart Buffer      8 ul
      DpnII(5U/ul)             2 ul
      BfaI (10U/ul)            4 ul
      H2O                     58 ul
      Total                   80 ul
                                    x 4
      gDNA (50ng/ul)           8 ul
      10x CutSmart Buffer      8 ul
      MspI (10U/ul)            4 ul
      H2O                     60 ul
      Total                   80 ul
      37C for 1 hour, 80C for 20min, 4C hold.

Step 2. Target capture

  • Reagents:
    • LMS_selector v2 probes (92nt; conc=1ng/ul, or 33nM)
    • Linker.V2 (stock primer 100uM; working tube 10uM): /5phos/GTTGGAGGCTCATCGTTATCCGACGGTAGTGTATGTTATCGAGGTCCGAC
    • Linker.V2_UMI (stock primer 100uM; working tube 10uM): /5phos/GTTGGAGGCTCATCGTTATCCGACGCCTATCGGGAAGCTGAAGNNNNNNNNNNGGTAGTGTATGTTATCGAGGTCCGAC
    • AmpLigase
    • Taq polymerase
  • Some adjustments:
    • Carefully match the probe:template ratio.
    • Test a gradient of annealing temperature (50C, 54C, 57C, 60C).
    • Test adding pre-annealed probe/linker duplex.
   Prepare probe/linker mix A (no UMI)
   LMS_selector v2 probes      20ul (probe:template=200:1)  
   Linker.V2 (100nM)           20ul (linker:probe=3:1)
   10X AmpLigase buffer         5ul
   Prepare probe/linker mix B (with UMI)
   LMS_selector v2 probes      20ul (probe:template=200:1)  
   Linker.V2_UMI (100nM)       20ul (linker:probe=3:1)
   10X AmpLigase buffer         5ul
   94C 2min -> 37C 10min


   Mix the MspI and DpnII/BfaI digested gDNA (20ul+20ul->40ul)
   Set up the following reactions, for annealing temperature there are six tubes: 
   1  w/o UMI, 50ng
   2  w/o UMI, 20ng
   3  w/o UMI, 0ng
   4  w/ UMI,  50ng
   5  w/ UMI,  20ng
   6  w/ UMI,  0ng
                                    x 8               x 8              x 8
   Mix digested gDNA           5ul               2ul             0ul
   10X AmpLigase buffer      1.5ul             1.8ul           1.8ul
   25mM MgCl2                1.6ul             1.6ul           1.6ul
   NAD (50mM)                0.4ul             0.4ul           0.4ul
   H2O                         7ul            12.2ul          14.2ul
   Total                    15.5ul              18ul            18ul
   94C 2min -> 50/52.8/57.4/60C, add 4.5ul/2ul/2ul pre-annealed probe/linker mix per tube 
       and incubate for 16-20 hours (started at 3pm on 11/24)
   -> add 1ul of 2.5U/ul Ampligase (1:1 dilution with H2O) to each tube (9am on 11/25)
   Note that I noticed some bubbles in many tubes, probably caused by the adding of probe/linker mix.
   So I briefly spin the tubes, and place them back to the thermal cycler. I decided to let the ligation 
   runs longer just in case the probes didn't anneal well with the template.
   -> 50/54/57/60C for 2 hour -> 94C for 2min -> 8C hold.
   -> add 2ul of Exo I/III mix -> 37C 2h -> 80C 20min.

   PCR amplification
   Circularization reaction      5 ul
   Kapa QPCR Master Mix         25 ul
   Linker.V2F (10uM)             1 ul
   Linker.V2R (10uM)             1 ul
   H2O                         9.0 ul
   Total                      25.0 ul
   95C 5min -> (95C 15sec -> 58C 30sec -> 72C 15 sec) x 30
   Monitor the amplification curves and terminate the reactions before plateauing.
  File:Selector 2014-11-25 50C.pngFile:Selector 2014-11-25 53C.png
  File:Selector 2014-11-25 57C.pngFile:Selector 2014-11-25 60C.png