VDS of the FRI
PCR Primer Design for pNIC-Bsa4 Cloning
Summer 11
Purpose: Design a forward and a reverse primer for PCR amplifying the CDS of your gene of interest sequence and
synthesizing Compatible Ends suitable for Ligation Independent Cloning (LIC) for insertion into the pNIC-Bsa4 as the
accepting vector for eventual expression.
You need to know the sequence of your Gene of Interest. You will only need to know the bases from the Start Codon to
the Stop Codon (the CDS – Coding DNA Sequence). We are not concerned about any flanking regions. Use the
codon-optimized DNA sequence from DNA Works and not the ‘true’ sequence from NCBI since the codonoptimized version is the one that your PCR will actually synthesize.
Refer to pNIC-Bsa4.pdf for information on how to design primers for LIC (Ligation Independent Cloning).
NOTE: You will be making the ‘Primers for LIC cloning’ and not the Sequencing Primers.
The Upstream and Downstream portions on this page represent the Leader and Lagger (or Prefix and Suffix) that
will be on the tail ends of your PCR product for cloning.
You will have to add 18-20 bp of your CDS onto the 3’ side of the Upstream bases.
You will have to add 18-20 bp of your CDS onto the 3’ side of the Downstream bases.
NOTE: Be sure to reverse complement the tail end of your CDS before adding the Downstream bases.
NOTE: The termination codon should be in either the Downstream Primer or in you Gene of Interest (GOI)
sequence – not both
Once you are satisfied with your primer sequence, Check for the GC content and Tm by going online to Analyze:
http://www.idtdna.com/analyzer/Applications/OligoAnalyzer/
Use 0.4 uM Oligo conc., 50 mM Na+, 0.3 mM dNTPs .
Test the Tm at 0, 1.5, 2, 4, and 6 mM Mg++ concentrations.
Write these values down because you will need them for PCR.
Post your results of these primers on the Wikispaces page for your Target (not your individual page)
save info into your lab notebook also.
Forward Primer:
5’ _______________________________________ 3’ ___ bp
GC Content ___%
0 mM Mg2+ Tm ___ oC 1.5 mM Mg2+ Tm ___ oC
2 mM Mg2+ Tm ___ oC
2+
o
2+
o
4 mM Mg Tm ___ C
6 mM Mg Tm ___ C
************************************************************************
Reverse Primer:
5’
_______________________________________
3’
Reverse complement it:
5’ _______________________________________ 3’ ___ bp
GC Content ___%
0 mM Mg2+ Tm ___ oC 1.5 mM Mg2+ Tm ___ oC
2 mM Mg2+ Tm ___
2+
o
2+
o
4 mM Mg Tm ___ C
6 mM Mg Tm ___ C
oC
************************************************************************
Create a virtual plasmid:
In WORD, paste
1. Insert: your CDS with Lagger and Leader portions of the primers added to the ends
2. Accepting Vector: full pNIC-Bsa4 sequence (FASTA sequence of pNIC-Bsa4 is on Google Docs)
In NEB Cutter online,
Virtually cut your Insert with BsaI to determine how the ends will be on your insert
Then in another NEB Cutter window, load the sequence of the Accepting Vector
Virtually cut your Accepting Vector with BsaI to determine where your GOI will fit inside the pNIC-Bsa4
sequence (HINT: use the zoom feature to see individual nucleotides)
Back in WORD - using these locations, paste together what your full plasmid should look like with the Gene inserted
Show this and highlight your gene and the start and stop codons.
Highlight where the 6xHIS tag is.
Save results of your Virtual Plasmid to the Google Docs (this part does not need to be on Wikispaces). Include
your Name and Date in the filename and on the document.