Daniel:Notebook/ComboLock/2017-1-23: Difference between revisions

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>Djacobse
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==Sample Matrix==
==Sample Matrix==


{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext>
|- style="background-color:#B1A0C7;font-size:12pt;font-weight:bold" align="center"
| width="65" height="30" | Tube No
| width="65" | Molecules Available
| width="65" | Amount


|- style="font-size:12pt" align="center"
| align="center" height="15" | 0 (AB)
| align="center" | 1.00E+11
| 1 pmol
|- style="background-color:#BFBFBF;font-size:12pt" align="center"
| align="center" height="15" | 2 (AB)
| align="center" | 1.00E+09
| 10 fmol
|- style="font-size:12pt" align="center"
| align="center" height="15" | 4 (AB)
| align="center" | 1.00E+07
| 100 amol
|- style="background-color:#BFBFBF;font-size:12pt" align="center"
| align="center" height="15" | 6 (AB)
| align="center" | 1.00E+05
| 1 amol
|}


==Protocol==
==Protocol==

Revision as of 23:48, 23 January 2017

Circularization Test

Back to Calendar

Since the results from the last experiment seemed to indicate that RCA hasn't been working for me, I'm going to try RCA again using the old positive amplicon systems. For examples see Oct-5-2016 and Sept-30-2016. I'll be using PCAmp4

Sample Matrix

Tube No Molecules Available Amount
0 (AB) 1.00E+11 1 pmol
2 (AB) 1.00E+09 10 fmol
4 (AB) 1.00E+07 100 amol
6 (AB) 1.00E+05 1 amol

Protocol

  1. Latch and Padlock Hybridization
    1. Suspend 2 uL (8 ug) beads per sample in 100 uL wash buffer
    2. Apply magnet for 30 sec and remove supernatant
    3. Add 2 uL oligo per sample to bead solution; incubate at RT for 5 min
    4. Apply magnet for 30 sec and remove supernatant
    5. Wash beads with 100 uL wash buffer; vortex to suspend; apply magnet and remove supernatant
    6. Add 2 uL per sample of 10 uM Padlock0501 to 20 uL 1X Amp ligase buffer
    7. Heat probes to 95C for 5 minutes
    8. Chill on ice for 3 minutes
    9. Add sample to prepared strep beads; vortex to suspend
    10. Incubate for 30 min at 37C with vigorous shaking
  2. Circularization
    1. Prepare 8.5X uL Phusion mix
    2. Reagent Stock Conc Final Amount 1x Vol (uL) MM Vol (8.5x) (uL)
      NAD+ 5 mM 40 nmol 8 68
      dNTP 1 mM 600 pmol 0.6 5.1
      Betaine 5 M 15 umol 3 25.5
      10X AmpLigase Buffer 10X 1X 2 17
      Amp Ligase 5 U/uL 10 U 2 17
      Phusion HF DNA Polymerase 2000 U/mL 6.4U 3.2 27.2
      nf H2O     1.2 8.4
      Total     20 140
    3. Add 20 uL phusion mix to 20 uL reaction
    4. Incubate at 55C for 4 hours