A. General Rules
1. Length:
Primers should contain at least 18 Ð 20 nucleotides complementary sequence:
to minimize the chances of encountering problems with secondary hybridization sites on the vector or insert
In the case of a sequencing primer, these 18-20 nt corresponding to the template sequence is all you need.
In the case of a cloning primer, these 18 Ð20 nt correspond to the part of the primer which is homologous to the template.
2.
Primers with long runs
of a single base should generally be avoided.
It is especially important to avoid 3 or more GÕs or
CÕs in a row (GGGCCC)
3.
Primers should have a
GC-content between 50 and 70 %
For primers which have a GC-content less than 50% it may be necessary to extend the primer sequence beyond 18 bases (corresponding to the template sequence).
4.
If possible, primers
should be stickier at the 5Õ ends than at the 3Õend.
Only one G or C at the 3Õend
5.
Primers should not
contain complimentary sequences (palindromes) within themselves. That is
they should not form hairpins. If this state exists, the primer will fold back
on itself and result in an unproductive priming event which decreases the
overall signal.
6.
Avoid Primer-Dimer
formation.
Primers should not contain sequences that would allow one primer molecule to anneal to another or to the other primer used in the sequencing reaction.
7.
Run Amplify to make sure
the primers really anneal to the target (i.e. are correct) and exclude the
possibility of primer-dimers.
8.
Check Tm is above 55¡C: http://micro.nwfsc.noaa.gov/protocols/oligoTMcalc.html
Tm of the two primers should be as close as possible to each other, max
2-3¡C apart.
9.
For Mutagenesis primers:
use PCR primers (RP) (reverse Phase purification).
This takes 3 days longer and costs more.
For all other primers: Desalted is pure enough!
10.
Order Primers by Fax or
on the Web with Invitrogen. Follow instructions in the Invitrogen booklet in
the lab. Smallest scale.
B. Example
Cloning
primer to PCR-clone Protein X into the vector pGEX-4T1
Protein X ORF sequence: atggggcgaggcagcggcacÉÉÉ..agctcatctcctttgactga
Stop and start codons in bold
Multiple cloning site of pGEX4T1 (a GST-tagging vector)
918/221
948/231
ctg gtt
ccg cgt gga tcc ccg gaa ttc ccg ggt cga ctc gag cgg ccg cat cgt
gac tga
leu val
pro arg gly ser pro glu phe pro gly arg leu glu arg pro his arg asp OPA
BamHI EcoRI
1) Check whether BamHI and EcoRI are absent in the ORF of proteinX
Only if this is the case, can these sites be used for cloning.
2) The forward primer corresponds to the sequence of the sense strand,
i.e. the ORF of ProteinX + restriction-enzyme sequence:
IMPORTANT: If cloning into a N-terminally tagged vector, the FRAME has to be respected! The start codon may be omitted in this case.
If cloning into a non-tagged or a C-terminally tagged vector, a Kozak-sequence (GCC GCC) has to be added before the ATG.
Two bases are added to the 5Õ-end
5Õ-GA GAA TTC atg ggg cga ggc agc ggc-3Õ
GC-content: 65%
Tm: 66¡C
3) The reverse primer corresponds to the reverse complement sequence of the
ORF, i.e. to the antisense strand and terminates in the stop-codon + restriction enzyme sequence
Write out the sense strand sequence to which this primer should be complementary to: 5Õ-ctcatctcctttgactga-3Õ
Now add the restriction site: 5Õ-ctcatctcctttgactgaGGATCC-3Õ
Write the corresponding antisense strand: 3Õ-gagtagaggaaactgactCCTAGG-5Õ
Reverse the sequence so that you are now reading 5Õ to 3Õ
5Õ-GGATCCtcagtcaaaggagatgag-3Õ
(in DNA-strider, this can be done by choosing ÒcomplementÓ and then ÒreverseÓ)
Add two random bases at the 5Õ-end
5Õ-GA GGATCCtcagtcaaaggagatgag-3Õ
GC-content: 50
Tm: 60¡C
To bring the Tm closer to the Tm of the Forward primer, add further complementary bases:
5Õ-GA GGATCCtcagtcaaaggagatgagctg-3Õ
GC: 52% Tm: 63¡C
Reduce Tm of forward primer by replacing G for A in the non-complementary region:
5Õ-AA GAA TTC atg ggg cga ggc agc ggc-3Õ
GC: 62% Tm: 64¡C
Check primers with Amplify and modify if necessary
Name primers: Initials-Protein-For and Initial-Protein-Rev
Example: SU-Hrs-For and SU-Hrs-Rev