Thread: Primer and PCR

pBR322 sequence with EcoRI cloning site GAATTC is identified in brackets. Primers are identified in brackets too

TCTCATGAGC GGATACATAT [primer 1]TTGAATGTAT TTAGAAAAAT[primer 1] AAACAAATAG GGGTTCCGCG
CACATTTCCC CGAAAAGTGC [primer 2]CACCTGACGT CTAAGAAACC] ATTATTATCA TGACATTAAC
CTATAAAAAT AGGCGTATCA [primer 3]CGAGGCCCTT TCGTCTTCAA[/size] [EcoRI cloning site]GAATTC[EcoRI cloning site] TCAT GTTTGACAGC
TTATCATCGA TAAGCTTTAA TGCGGTAGTT TATCACAGTT AAATTGCTAA CGCAGTCAGG
CACCGTGTAT GAAATCTAAC [primer 4]AATGCGCTCA TCGTCATCCT[/size] CGGCACCGTC ACCCTGGATG

4 primers - 2 reverse and 2 forward

I have identified primer 1 and 2 as forwards and primer 3 and 4` as reverse

Primer 1: 5’ TTGAATGTATTTAGAAAAAT 3’
Primer 2: 5’ CGAAAAGTGCCACCTGACGT 3’
Primer 3: 5’ TTGAAGACGAAAGGGCCTCG 3’
Primer 4: 5’ AGGATGACGATGAGCGCATT 3’


If I used this plasmid to code a 100bp cDNA fragment what would the size of my cDNA be if I use it as a template (where do I start and begin)

and how on earth do I use 2 of the primers above to amplify a PCR plasmid sequence containing the 100 bp CDNA as well as a third primer as an internal primer

from what I gather the rules concerning primers are that GC rich areas and multiple sites are a NO NO which all my primers have and I take the 5' position of the forward primer and the 3' position of the reverse primer to make the internal primer. Am I missing any rules here?

Help