Dinh 2011/NOTES/2011-8-30: Difference between revisions
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TBU-gel | TBU-gel | ||
[[File:DMR330K-E circularization with lambda exo.jpg]] | [[File:DMR330K-E circularization with lambda exo.jpg]] | ||
A - 10 bp ladder (1ul) | A - 10 bp ladder (1ul) | ||
B - Linear ssDNA probes (78ng) | B - Linear ssDNA probes (78ng) | ||
C - Purified circle probes (1.3ng/ulx10ul = 13ng) | C - Purified circle probes (1.3ng/ulx10ul = 13ng) | ||
===Test circularization #2: Changing the amount of splint.=== | ===Test circularization #2: Changing the amount of splint.=== |
Latest revision as of 06:02, 31 August 2011
Barcoding DMR330K probes[edit]
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[edit]
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.[edit]
- 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: