Download SMART cDNA Library Construction Kit User Manual

SMART TM cDNA Library Construction Kit User Manual
VIII.Troubleshooting Guide
Obtain or isolate
total or poly A+ RNA
(Section IV.B)
Recommended: Check quality
of RNA on a gel (Section IV.B)
Synthesize first-strand cDNA
(Section V.A & VI.A)
Troubleshooting:
see Section VIII.A & B
ds cDNA synthesis by LD PCR or
by Primer Extension
Check quality of ds cDNA
product on a gel
Proceed to next step
if results normal
Repeat Section IV.B
if necessary
Repeat Sections IV.B,
V.A, or VI.A if necessary;
check stability of RNA
Repeat Sections IV.B, V.A,
or V.B if necessary;
check stability of RNA
If undercycled, incubate for 3–5
more cycles and recheck product
If overcycled, repeat Section V.B
with a fresh aliquot of first-strand
DNA using fewer cycles
For Primer extension
troubleshooting, see Section VIII.F
Sfi I digestion
cDNA size fractionation
Check fractions on an
agarose gel
Pool fractions containing cDNA > 400 bp
Ligate ds cDNA to λ arms and
package (Section VII.A)
PCR Screening of cDNA insert
(Optional)
Check titer and % recombinants of
library (Sections VII.C & D)
Troubleshooting:
see Sections VIII.H & I
Figure 5. Guide to troubleshooting SMART cDNA synthesis. Please refer to the text for further
information on troubleshooting procedures.
Protocol No. PT3000-1
www.clontech.com
Version No. PR7Y2399
Clontech Laboratories, Inc.
37