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Developing
Primers using DNASTAR software 1.
Go to DNASTAR under Programs and select SeqMan. 2.
Select Add to New. Select all
of the exported lanes. 3.
Set the vector as pUC19HindIII (restriction site is AAGCTT). 4.
Assemble. Program will
assemble sequences into related contigs. 5.
Go to Contig, then Save Consensus. Save
the consensus as library number, contig number (e.g. lib 4, contig 1). 6.
Go to DNASTAR under Programs and select PRIMERSELECT. 7.
Click on File, then Enter Sequence. 8.
Individually select the consensus file. 9.
Find the repeat and highlight. Click
on Conditions, then Primer Location, then select Upper & Lower.
Go one repeat in on both sides. 10.
Start with default settings for Primer Characteristics. Change
if no primer pairs found with those settings. 11.
Click on Locate, then Primers & Probes. 12.
Click on Locate, then PCR Primer Pairs. 13.
When selecting a primer pair, dTm should be less than 5.
Resulting product should be greater than 120 bases, but less than
250. Examine score.
Under Report, it should be less than –5 for worst dimer.
Delta G should drop toward 3’ end. 14.
Double-click on selected primer pair.
Click on Conditions, then Lock both primers. 15. Print amplification summary. Label printout and save file with lib #, contig #. |
Last Updated: 11/04/02
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