Biology Reference
In-Depth Information
3.7 Purify
Immunoprecipitated
Chromatin
a. Recover DNA by phenol-chloroform extraction and ethanol
precipitation. (Optional: total yield of DNA from ChIP is very
low, it might be very hard to locate the DNA pellet after etha-
nol precipitation. Addition of 20
μ
g glycogen carrier helps visu-
alize the DNA pellet).
b. Wash pellets with 70 % ethanol and air dry.
c. Resuspend DNA in 50
μ
L TE.
3.8 Real-Time PCR
(qPCR) ( See Notes 4
and 5 )
In this context, we use DyNAmo™ Hot Start SYBR
®
Green qPCR
Kit (Thermo Scientifi c) as a tool to quantitatively measure the
immunoprecipitated viral genome on an ABI 7500 real-time PCR
machine. Any other SYBR green qPCR kits and real-time PCR
machine could be used according to manufacturer's instructions.
Use PCR-grade tubes, DNase/RNase-free barrier pipet tips, and
DNase/RNase-free ultrapure water for all of the following steps.
Always include control reactions: negative control with primers but
no input DNA and a series of positive controls as a standard curve.
1. Prepare standard curve DNA. Dilute 1.02
μ
g Ad plasmid
DNA pTG3602 with ddH
2
O to 500
μ
L to obtain 10
8
cop-
ies/2
l stock (
see
Note 6
). Then perform tenfold serial dilu-
tions in ddH
2
O ranging from 10
8
copies down to 1 copy/2
μ
μ
L.
L of standard DNA in each reaction.
2. Prepare 2× SYBR green master mix. Add 12
Use 2
μ
L 50× Rox into
every 1 mL SYBR green master mix, mix well by vortexing.
This mixture will serve as a 2× master mix in the next step.
Rox is a passive reference dye provided along with SYBR green
qPCR kit. The concentration of Rox is vary depending on the
model of real-time PCR machine, please refer to manufactur-
er's instruction before use.
3. Prepare qPCR reaction mix in a proper size tube (Table
1
).
To ensure the precision of qPCR reaction, every sample,
including standard DNA and negative control, should be run
in three replicates. Calculate total number of reactions needed,
and always prepare 5 % extra reactions.
μ
Table 1
Reaction setup for ABI qPCR instruments
Component
Volume
Final concentration
2× SYBR green master mix
10
μ
L
1×
Forward primer (10 mM)
1
μ
L
0.5
μ
M
Reverse primer (10 mM)
1
μ
L
0.5
μ
M
ddH
2
O
6
μ
L
μ
L/reaction
a
a
Template DNA is not included
Total volume 18