32P-cDNA SYNTHESIS

●●●●●　³²P-cDNA SYNTHESIS　●●●●●

　32P labeled single-stranded cDNA (sscDNA) can be used in a variety of techniques; as a hybridization probe, for renaturation studies, or for eventual cloning. The most important factor in synthesizing cDNA is that the mRNA template be as pure as possible - free of RNase, rRNA, inhibitory compounds (especially polysaccharides), and, if a specific cDNA is desired, extraineous poly(A)-containing RNAs. This is a technically difficult technique, so don't be discouraged if it doesn't work the first couple of times. If it doesn't work, retest the poly(A)-containing RNA by agarose-urea electrophoresis to see if it is still intact.
　Strict RNase-free technique should be used throughout this procedure. All glassware, plasticware, and buffers should be autoclaved, except for the dNTPs, 2-ME, and oligo(dT)12-18.

10X cDNA buffer (per 100ml)

	final conc.
6.06 grams Tris-OH	0.5M
2.03 grams MgCl2-7H2O	0.1M
10.44 grams KCl	1.4M

Adjust to pH8.3 at 42C with conc. HCl

0.3M 2-mercaptoethanol
　131ul 2-ME plus 4.87ml ddH2O

20mM dNTPs

		ddH2O
dATP	25mg	2.15ml
dCTP	25mg	2.35ml
dTTP	25mg	2.23ml
dGTP	25mg	2.12ml

Add ddH2O directly to the bottle, dissolve, and adjust to neutrality with 50mM Tris, pH7

TEN (per liter)

		final
1.2114 grams	Tris-OH, pH 7.8	10mM
0.5844 grams	NaCl	10mM
0.3802 grams	Na4EDTA	1mM

8u/ml oligo(dT)12-18
　Add 125ul ddH2O/A260 unit directly to the vial

Transfer 50uCi alpha³²P-dCTP to a 500ul microfuge tube, plug it with cotton, and cover it with a double layer of parafilm. Puncture the parafilm several times with a needle.

Dry in a vacuum dessicator under vacuum to complete dryness (usually 15-30 min.). Remove and discard the parafilm and cotton.

Working on ice, add the following reagents to the vial:

5ul	10X cDNA buffer
5ul	0.3M 2-ME
5ul	8u/ml oligo(dT)12-18
2.5ul	@ 20mM dATP, dGTP, and dTTP
1ul	20mM dCTP
5ul	1ug/ul poly(A)-containing RNA
14.25ul	ddH2O

Vortex, and add 7.25ul reverse transcriptase (50 units). Gently vortex, spin 5 sec., and incubate for 1 hr at 42C.

Drop in a boiling water bath for 2 min., and rapidly cool on ice. Centrifuge for 1 min., and transfer to a fresh microfuge tube. Store at -20C.

To determine incorporation, dilute a 2.5ul aliquot of sscDNA with 40ul TEN. Add 5ul glycerol, and vortex.

Apply this sample to a ~0.5 x 30cm sephadex G50(50-150) column, equilibrated with TEN. Collect 32 0.5ml fractions into 6ml scintillation vials containing 5ml scintillation fluid each.

Cap and mix the vials, and count them by scintillation spectrophotometry.

CALCULATIONS

incorporated CPM = (cDNA peak - background) x 20

ng cDNA = (cDNA peak - background)/(total CPM - background)x 4 bases x 6600 ng dNMP

NOTES

* If 32P is needed only to trace the reaction, and not for later autoradiography, then decrease the 32P-dCTP to 5uCi and add 2.5ul dCTP rather than just 1ul. Under these conditions;

ng cDNA = (cDNA peak - background)/(total CPM - background)x 4 bases x 16500 ng dNMP

* If you desire or need to use an RNase inhibitor, then replace the 2-ME with 2.5ul 0.1M DTT and add 5 units RNasin and 100ug/ml BSA.

* Final reaction mixture:

50mM	Tris, pH 8.3 (at 42C)
10mM	MgCl2
140mM	KCl
30mM	2-ME
0.04 A260 units	oligo(dT)12-18
1mM @	dATP, dGTP, and dTTP
0.4mM	(50uCi) 32P-dCTP
5ug	poly(A)-containing RNA
50 units	reverse transcriptase
50ul total volume

Rosemarie Spencer (personal communication)
Buell, et al. 1978 J. Biol. Chem. 253:2471-2482
Wickens, et al. 1978 J. Biol. Chem. 253:2483-2495

Other references:
Land, et al. 1981 Nucl. Acids Res. 9(10):2251
J. Vaughn (personal communication)
BRL Product Profile: AMV Reverse transcriptase
Monahan, et al. 1976 Biochemistry 15:223-233
Baulcombe and Verma 1978 Nucl. Acids Res. 5(11):4141