AlanFung:LabNotes/CTCF/2010-1-15: Difference between revisions

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*Only 50ng,10ng, 5ng was amplified based on the QPCR curve, will run a gel to quantify

Revision as of 17:53, 19 January 2010

Quantify Bisulfite Converted Samples with Qubit ssDNA Kit

On 50ng, 10ng, 5ng, 1ng, 0.5ng, 0.1ng & 0.05ng P21-200BP

7 samples
Prepare 7 Qubit Assay tubes

Prepare 7*200ul diluted dye=1400ul
Mix 7ul dye with 1393ul buffer
Mix 2ul samples with 98ul diluted dye
vortex spin down and wait for 2 mins

Result
Start (ng) Sample Concentration ng/ul Volume Amount Recovered (ng) Yield %
50 1.7 20 34 68
10 0.25 20 5 50
5 0.13 20 2.6 52
1 0.26 20 5.2 520
0.5 N/A 20
0.1 N/A 20
0.05 N/A 20

PCR quantification of Bisulfite treated samples using 3 paris of CHR bilsulfite conversion primers

On 50ng, 10ng, 5ng, 1ng, 0.5ng, 0.1ng & 0.05ng P21-200BP

1 sample CHR22 CHR21 CHR8
2X iQ Super Mix 20 20 20
Primer F (3.3uM) 6 6 6
Primer R (3.3uM) 6 6 6
7 Samples CHR22 CHR21 CHR8
2X iQ Super Mix 160 160 160
Primer F (3.3uM) 48 48 48
Primer R (3.3uM) 48 48 48
Setup
CHR22 CHR21 CHR8
1 50ng 50ng 50ng
2 10ng 10ng 10ng
3 5ng 5ng 5ng
4 1ng 1ng 1ng
5 0.5ng 0.5ng 0.5ng
6 0.1ng 0.1ng 0.1ng
7 0.05ng 0.05ng 0.05ng
  • Only 50ng,10ng, 5ng was amplified based on the QPCR curve, will run a gel to quantify