C DNA
C DNA
C DNA
Good luck.
Narendra Kaushik
Neuroscience & Psycological Medicine,
Imperial College at Charing Cross Hospital,
Fulham Palace Road, London W6 8RF UK
email. nkaushik at hgmp.mrc.ac.uk
Final volume 20 ul
dH2O X ul
5X buffer 4.0 ul
DTT ( 0.1 M) 2.0 ul
dNTP (10mM) 1.0 ul
mRNA 0.5 ug (on the beads).
Total 19.0 ul
SupersriptII 1.5 ul (Life technologies).
Put the tube into the magnetic strand and discard the buffer, 2x wash
with TE (10mM Tris pH 8.0, 1mM EDTA).
A-Tailing.
PCR.
use 50ng of mRNA equivalent beads in 100ul reaction for PCR.
94oC/5min x1
94oC/30s
60oC/2min
72oC/4min
25 cycles
72oC/10min
Dont use too many cycles to amplify.
Size select by purifying fron agarose gel(1.5%, TAE) > 500bp and
clone into the vector
Comparison of Reaction Assemblies and Protocols
Water is usually treated with 0.1% v/v diethylpyrocarbonate for at least 1 hour at 37°C
and then autoclaved (at least 15 min) to inactivate traces of DEPC. Inactivation of DEPC
in this manner yields CO2, H2O and EtOH. Higher concentrations of DEPC are competent
of deactivating larger amounts of RNAse but remaining traces or byproducts may inhibit
further biochemical reactions such as in vitro translation. Further on, chemical
modification of RNA such as carboxymethylation is possible when traces of DEPC or its
byproducts are present, resulting to reduced usage of RNA even after buffer exchange
(after precipitation).
DEPC treated (and therefore RNase-free) water is used in handling of RNA in the
laboratory, to reduce the risk of RNA being degraded by RNases.
DEPC derivatization of histidines is also used to study the importance of histidyl residues
in enzymes. Modification of histidine by DEPC results in carbethoxylate derivates at the
N-omega-2 nitrogen of theimidazole ring. DEPC modification of histidines can be
reversed by treatment with 0.5M hydroxylamineat neutral pH