SNAPSHOT TIPS

TEMPLATE TIPS

Remove unincorporated dNTPs and PCR primers with Exo-SAP

Template should be 0.01-0.4 pmol purified PCR product FOR EACH template- primer pair (not combined total)

Unlabeled primer

Tm (not including tail) >50*C

Minimum length 18-25 bp

HOWEVER, less than 36 bp fragment mobility is not just length dependent (dyes and base composition pay a role). Markers must be separated by at least 6 bp
Primers above 36 bp migrate according to length only. Markers must be separated by at least 4 bp
If too long, increase chance of secondary structure and increased TM
5’ tail
Tails must be non-Homologous
Poly T, C or GACT
NOT poly A
Check for homology to template, primer dimer, and secondary structures
For multiplexing - each tail is a different length (at least 4-8 bases apart)
Not multiplexing – do not need a tail

HPLC purification recommended for primers longer than 30 bp (double HPLC for primers >60 bp)

Size end points for analysis 25-110 bp

0.2 ml PCR tube

Template and primer mixed to a total volume of 5 ul

Can multipex up to 10 primers