Sequencing of clones from 2H screens


The insert of isolated clones from two-hybrid screens can be sequenced after PCR amplification of the gene in the pJG plasmid. Iti is not necessary to isolate the corresponding plasmid by transformation of E.coli. The insert of the pJG plasmid is amplified by PCR with primers on the 5' and 3' to the cloning site from total yeast DNA. After phenol extraction and precipitation by ethanol, the DNA is sequenced with a primer 40 bp 5' to the EcoRI site. It is usually not necessary to purify the PCR band by gel electrophoresis and quia-quick extraction from the agarose.

nnn µl  H2O
5 µl   PG8+PG7 (each 10 µM)
5 µl   10x PCR buffer
1 µl    50x dNTP mix (each 10mM)
1 U    Taq
1 µl   yeast DNA with JG plasmid
------
50 µl

Programme:
2 min  94°C (only once)
30 s     94°C
1 min   55°C
3 min   72°C  (or longer, if necessary)
repeat 35x

  • check PCR on a agarose gel:  5 µl for each reaction
  • you may dilute the reactions to 100 µl with TE
  • extract PCR reaction with phenol:chloroform and with chloroform
  • precipitate with 2,5 volumes of EtOH
  • wash with 70% EtOH, dry in speed-vac
  • dissolve in 10 µl water (store at -20°C)
  • use specified amount for sequencing
  • sequence with oligo PG6, 300 bp are sufficient
Oligos:

PG8:  GAC TGG CTG AAA TCG AAT GGT
PG7:  AGA AAT TCG CTT ATT TAG AAG
PG6:  TGC TGA GTG GAG ATG CCT CC