Dinh 2011/NOTES/2011-8-30

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Revision as of 00:01, 31 August 2011 by >Dinh
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Barcoding DMR330K probes

New primers:

Sequence Name Sequence
pAP1V61U_w 5’-/5Phos/GGGTCATATCGGTCACTGTU-3’
pAP2V6_s 5’-/5Phos/C*A*C*GGGTAGTGTGTATCCTG-3’

Noi amplified and purified 2x32 reactions of DMR330K-E mixed probes:

Tube 1 = 105ng/ul x 210 ul = 22 ug
Tube 2 = 89ng/ul x 210 ul = 18.7 ug

Lambda Exo:

DMR330K amplicons ~20ug, 210 ul
Lambda exo buffer (10x),  25 ul
Lambda exo                10 ul
H2O                        5 ul
===============================
                         250 ul
Purified with 2 columns for each tube.

Digested Tube 1 with Lambda exo for 1 hour, yield: 79ng/ul x 70 ul = 5.53 ug
Digested Tube 2 with Lambda exo for 1 hour, yield: 98ng/ul x 70 ul = 6.86 ug

Test circularization #1: previous conditions

Reaction:
Lambda digested probes (79ng/ul, 5ug)    64 ul
APV6_splint (100uM)                     2.2 ul
Ampligase buffer (10x)                    8 ul
H2O                                     5.8 ul
================================================
                                         80 ul
Program:
95C 30s -> 94.8C 30s -> (-0.2C ) x 199 cycles -> 
55C forever (add 9ul Ampligase + 1ul buffer) -> 
(55C 2 min -> 94C 30s) x 12 cycles -> 55C forever -> 
37C 2min (add 2.5 ul ExoI, 2.5 ul Exo III, 1 ul ExoIII buffer, 4 ul H20) -> 37C 1 hr -> 90C 5min -> 4C forever

Purified with column, elute in 50ul EB, yield: 1.3ng/ul x 50 = 65 ng

TBU-gel File:DMR330K-E circularization with lambda exo.jpg A - 10 bp ladder (1ul) B - Linear ssDNA probes (78ng) C - Purified circle probes (1.3ng/ulx10ul = 13ng)

Test circularization #2: Changing the amount of splint.

  • Amount of splint to probes now changed because targets are single-stranded.
Reaction:
Lambda digested probes, 0.5 ug          6.3 ul (79ng/ul)
APV6_splint + H2O (10uM)          
           8:1                    2.2 + 0.5 ul
           4:1                    1.1 + 1.6 ul
           2:1                    0.6 + 2.1 ul
Ampligase buffer (10x)                    1 ul
================================================
                                         10 ul
Program:
95C 30s -> 94.8C 30s -> (-0.2C ) x 199 cycles -> at 59C add 3 ul of (3 ul Ampligase, 1 ul Ampligase buffer, and 6 ul H2O) ->
55C forever -> 
(55C 2 min -> 94C 30s) x 40 cycles -> 55C forever -> 4C forever *skip ExoI/III 

Run TBU gel: