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cDNA help need

Wenjun Huang biocwjh at osuvm1.bitnet
Thu Dec 8 00:30:11 EST 1994


	I need a help! (for my first strand cDNA synthesis from cotton cell
suspension culture)
	The total RNA was isolated by use of extraction buffer (containing Tris,
NaCl, EDTA and ATA), phenol/chloroform, LiCl precipitation,
phenol/chloroform, ethanol/NHAc precipitation.  The ratio of A260/280 of
the RNA is 1.9-2.0, the RNA yield is about 600 mg/10 g cells.
	The poly(A)+RNA was selected by oligo(dT) cellulose column (GIBCO BRL,
ultra PURETM).  The ratio of A260/280  of the selected poly(A)+RNA is
1.98-2.02.  The RNA and poly(A)+RNA looked fine based on formaldehyde gel
electrophoresis and in vitro translation.  
	I used ZAP-cDNA synthesis kit (Stratagene) to construct the cotton cDNA
library.  However, I did not get the first strand cDNA.  Strangely, when
I continued the second strand cDNA synthesis, I got smear band with
reasonable sizes on developed X-ray film.  The test control RNA provided
by the kit always worked.  Did my sample poly(A)+RNA degrade during cDNA
synthesis? I added reaction buffer into sample poly(A)+RNA and incubated,
then ran formaldehyde gel, the poly(A)+RNA was still there with a little
bit degradation.   
	Finally, I did first strand cDNA synthesis in this way:  to Tube A,
added 0.2 mg of test control RNA; to Tube B, added 0.5 mg of sample
poly(A)+RNA; to Tube C, added 0.25 mg of sample poly(A)+RNA and 0.1 mg of
test control RNA; to Tube D, added 1 mg of sample total RNA and 0.1 mg of
test control RNA.  Tube A gave a 1.8 Kb control band and Tube D gave a
smear band containing a 1.8 Kb control band, Tube B & C showed nothing!  
	 I guess that my sample poly(A)+RNA get problems.  If those people who
have suggestions, explanations and successful protocols, please tell me. 
Thank you in advance.



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