Used for cloning of unknown 5’-ends (provided internal sequence is available for primer design)
cut out the extension product and slice it into small pieces
add 100 ul of 0.5 M NH4oAc, 10 mM MgCl; 0.1% SDS
incubate with shaking overnight at RT
spin down the acrylamide and wash with 100 ul of the same buffer
transfer solution in 0.5 ml tube and extract solution with 200 ul of phenol/chl x2
extract with 400 ul of butanol, add 5 mg oh glycogen
precipitate with 3V of ethanol at -80
wash the pellet with 70% ethanol
dissolve the pellet in 25 ul of water
mix 3 ul of 25 mM cobalt solution (supplied with the enzyme, Boehringer) with 2 ul of water; it gives 15 mM 10x stock
mix in the following order:
5x buffer 4 ul
10X Co 2 ul
1 mM dGTP 1 ul
cDNA in 12 ul
TdT, 20 u/ul 1 ul
incubate for 30 min at 37 C
add 20 ul of water and 1 ul of 10% SDS
extract with 50 ul of phenol/chl
add 3 ul of 3M NaoAc, pH 7.0
precipitate with 200 ul of ethanol, wash with 100% ethanol, resuspend in 20 ul of water
set up PCR in 50 ul using 1 ul of G-tailed cDNA with 0.5 mM of primers (one oligo[C] 15-mer, one- sequence- specific; 0.2 mM of each dNTP; 2.5 mM of MgCl; 94C-30; 65C-30; 72 C-1 min. 35 cycles. Take 5 ul for the gel.
If product is not visible or non-specific, take 1 ul for the second PCR. Same conditions. 35 cycles