Rui:LabNotes/Haplotyping/2014-12-2: Difference between revisions

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==Procedure==
==Procedure==
* 1a. R.E digestion
===1a. R.E digestion===
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8<\hiddentext>
|- style="background-color:#CCFFCC;font-size:10pt"
|style="font-weight:bold" width="192" height="15" | Components
| width="72" | 48ng
| width="72" | 24ng
| width="79" | 12ng
| width="79" | NTC
 
|- style="font-size:10pt"
| height="15" valign="bottom" | gDNA (12ng/ul)
| align="center" align="right" valign="bottom" | 4.00
| align="center" align="right" valign="bottom" | 2.00
| align="center" align="right" valign="bottom" | 1.00
| align="center" align="right" valign="bottom" | 0.00
 
|- style="font-size:10pt"
| height="15" | 10x cutsmart buffer
| align="center" align="right" valign="bottom" | 1.00
| align="center" align="right" valign="bottom" | 1.00
| align="center" align="right" valign="bottom" | 1.00
| align="center" align="right" valign="bottom" | 1.00
 
|- style="font-size:10pt"
|style="color:#FF0000" height="15" | DpnII (50U/ul) -> dilute to 5U/ul
| align="center" align="right" valign="bottom" | 2.50
| align="center" align="right" valign="bottom" | 1.25
| align="center" align="right" valign="bottom" | 0.63
| align="center" align="right" valign="bottom" | 0.63
 
|- style="font-size:10pt"
|style="color:#FF0000" height="15"  valign="bottom" | BfaI (10U/ul) -> dilute to 2U/ul
| align="center" align="right" valign="bottom" | 2.50
| align="center" align="right" valign="bottom" | 1.25
| align="center" align="right" valign="bottom" | 0.63
| align="center" align="right" valign="bottom" | 0.63
 
|- style="font-size:10pt"
| height="15"  valign="bottom" | H2O
| align="center" align="right" | 0.00
| align="center" align="right" | 4.50
| align="center" align="right" | 6.75
| align="center" align="right" | 7.75
 
|- style="font-size:10pt"
| height="15"  valign="bottom" | Total
| align="center" align="right" | 10.00
| align="center" align="right" | 10.00
| align="center" align="right" | 10.00
| align="center" align="right" | 10.00
 
|}
 
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8<\hiddentext>
|- style="background-color:#D9D9D9;font-size:10pt;font-weight:bold"
| width="192" height="15"  | Temperature
| width="72" align="center" valign="bottom" | Duration
 
|- style="font-size:10pt"
| height="15" | Digestion
| 37C, 1hr
 
|- style="font-size:10pt"
| height="15" | hold
| 4C
 
|}
 
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8<\hiddentext>
|- style="background-color:#D9D9D9;font-size:10pt;font-weight:bold"
| width="192" height="15"  | Components
| width="72" align="center" | Volume (ul)
| width="72" align="center" | Final conc.
 
|- style="font-size:10pt"
| height="15" valign="bottom" | 10X NEBuffer 3.1
| align="center" align="center" | 1
| align="center" | 1x
 
|}
 
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8<\hiddentext>
|- style="background-color:#D9D9D9;font-size:10pt;font-weight:bold"
| width="192" height="15"  | Temperature
| width="72" align="center" valign="bottom" | Duration
 
|- style="font-size:10pt"
| height="15" | Digestion
| 37C, 30min
 
|- style="font-size:10pt"  valign="bottom"
| height="15" | heat inactivation
| 80C, 20min
 
|- style="font-size:10pt"
| height="15" | hold
| 4C
 
|}
 
===1b. Assembly of selecotr and linker ===

Revision as of 08:32, 4 December 2014

Purpose

  • repeat selector procedure:
  • selector : gDNA = 200:1; linker : selector = 3:1
  • direct gDNA input: 12, 24, 48ng
  • compare selector vs padlock probe
  • premade

Samples

  • HapMap gDNA from Coriell GM18506 (diluted to 50ng/ul)
  gDNA input Selector set Probe ratio Indx Test
#1 48ng linkerV2_UMI 200:1; 3:1 Indx01 selector_UMI
#2 24ng linkerV2_UMI 200:1; 3:1 Indx02 selector_UMI
#3 12ng linkerV2_UMI 200:1; 3:1 Indx03 selector_UMI
#4 NTC linkerV2_UMI 200:1; 3:1 Indx04 selector_UMI
#5 48ng linkerV2 200:1; 3:1 Indx05 selector
#6 24ng linkerV2 200:1; 3:1 Indx06 selector
#7 12ng linkerV2 200:1; 3:1 Indx07 selector
#8 NTC linkerV2 200:1; 3:1 Indx08 selector
#9 48ng linkerV2 200:1 Indx09 padlock probe
#10 24ng linkerV2 200:1 Indx10 padlock probe
#11 12ng linkerV2 200:1 Indx11 padlock probe
#12 NTC linkerV2 200:1 Indx12 padlock probe
#13 50ng_digested (Kun's, asPC) linkerV2_UMI 200:1; 3:1 Indx13 selector_UMI
#14 20ng_digested (Kun's, asPC) linkerV2_UMI 200:1; 3:1 Indx14 selector_UMI

Procedure

1a. R.E digestion

Components 48ng 24ng 12ng NTC
gDNA (12ng/ul) 4.00 2.00 1.00 0.00
10x cutsmart buffer 1.00 1.00 1.00 1.00
DpnII (50U/ul) -> dilute to 5U/ul 2.50 1.25 0.63 0.63
BfaI (10U/ul) -> dilute to 2U/ul 2.50 1.25 0.63 0.63
H2O 0.00 4.50 6.75 7.75
Total 10.00 10.00 10.00 10.00
Temperature Duration
Digestion 37C, 1hr
hold 4C
Components Volume (ul) Final conc.
10X NEBuffer 3.1 1 1x
Temperature Duration
Digestion 37C, 30min
heat inactivation 80C, 20min
hold 4C

1b. Assembly of selecotr and linker