Copy of Primer Design Guidelines for PCR Cloning

Basic Process for Designing Primers for Cloning

Locate your template
1. For example, suppose we want to clone the sequence for pLtetO: tccctatcagtgatagagattgacatccctatcagtgatagagatactgagcacatcagcaggacgcactgacc
Design a REVERSE primer with the basic structure 5' BUFFER NUCLEOTIDES - RESTRICTION SITE - TEMPLATE ANNEALING PORTION 3'
1. TEMPLATE ANNEALING PORTION
  1. Since this the FORWARD primer, simply select the 5' nucleotides of the template. For example, tccctatcagtgatagagattgacatc in the pLtetO example.
  2. Use a melting temperature calculator to determine the Nearest Neighbor melting temperature (TmNN). There are different ways of calculating melting temperatures but this is often the most accurate. I like to use http://www.basic.northwestern.edu/biotools/oligocalc.html. For example, tccctatcagtgatagagattgacatc in the pLtetO example has a TmNN of 56.23C. #### Another option is to calculate Tm using the IDT Oligo Analyzer: http://www.idtdna.com/analyzer/applications/oligoanalyzer/##### IDT recommends using the following settings, which are similar to the mastermix in most PCR reacitons:###### Oligo Concentration: .25 uM
    1. 1. Na+ Concentration: 50 mM
        
        Mg++ Concentration: 3 mM
        
        dNTPs Concentration: .8 mM
  3. Aim for TmNN of >= 50C. The more bases you choose for this portion, the greater the TmNN will be. I usually go from 50-53C and not higher, since it is often not necessary and just adds more cost and complexity to oligo synthesis.
  4. That being said, in the pLtetO example above, we selected a longer template annealing portion with a higher TmNN because there is an internal repeat in the pLtetO sequence (see the two bolded and underlined regions which are exactly the same in the sequence tccctatcagtgatagagattgacatccctatcagtgatagagatactgagcacatcagcaggacgcactgacc). Thus, in order to get specific binding of the primer to the template, we want additional length to the primer. Remember that PCR extension occurs at the 3' end of the primer. Thus, we want to ensure that there is no nonspecific binding at the 3' end of your designed primer. Thus, we designed our template annealing portion to include additional base pairs so that the 3' end of the template annealing portion can only bind in one way to the template.
2. RESTRICTION SITE
  1. Check the NEB website (http://www.neb.com) for your restriction site sequence. For example, MluI is ACGCGT. Note that many restriction sites are palindromes.
  2. Check your template and the vector you will be cloning into to make sure that it does not contain the restriction site that you want. You can do this using many different programs. One good web-based tool is http://tools.neb.com/NEBcutter2/index.php
3. BUFFER NUCLEOTIDES
  1. These are nucleotides that must be added upstream (to the 5' end of the restriction site) for efficient cleavage. NEB has information on how many upstream sites you need to add for efficient cleavage, which you can find at http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/cleavage_linearized_vector.asp and http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/cleavage_olignucleotides.asp. The first link is often more accurate because it describes the case where you have a restriction enzyme at the 5' end of a relatively long piece of DNA. However, in some cases where you are cutting short DNA such as oligos, the second link may be more accurate. For example, the NEB Website states that MluI is cleaved with 99% efficiency with 2 BUFFER NUCLEOTIDES (let's choose 5' CT 3' randomly for our BUFFER NUCLEOTIDES).
  2. By default, if you don't find information on the links above, you should select about 6 nucleotides for your BUFFER NUCLEOTIDES.
4. ASSEMBLE AND NAME THE SEQUENCE
  1. For example, for MluI-pLtetO-f, we assemble 5' CT ACGCGT tccctatcagtgatagagattgacatc 3'
  2. Calculate the total TmNN of the entire primer. In this case, it is 65C.
  3. Record the TmNN of the total primer and the TmNN of the TEMPLATE ANNEALING PORTION (e.g., TmNN 65C/56C)
  4. Choose a descriptive name for the primer. I like the convention of listing the restriction enzyme names, the template names, and the direction. For example, MluI-pLtetO-f states that this primer contains (from 5' to 3'), an MluI RESTRICTION SITE followed by a TEMPLATE ANNEALING PORTION which is in the forward direction of the template.
  5. So I would record this primer as 5' CT ACGCGT tccctatcagtgatagagattgacatc 3' MluI-pLtetO-f TmNN 65C/56C.
Design a REVERSE primer with the basic structure 5' BUFFER NUCLEOTIDES - RESTRICTION SITE - TEMPLATE ANNEALING PORTION 3'
1. TEMPLATE ANNEALING PORTION
  1. Select a TEMPLATE ANNEALING PORTION with TmNN that matches the TEMPLATE ANNEALING PORTION of your FORWARD PRIMEr. For example, the TEMPLATE ANNEALING PORTION TmNN of the FORWARD PRIMER MluI-pLtetO-f is 56C. The sequence in pLtetO atcagcaggacgcactgacc has a TmNN of 56C also and is thus a suitable target for designing the TEMPLATE ANNEALING PORTION of the REVERSE PRIMER.
  2. You must remember to properly reverse complement the template target to get the TEMPLATE ANNEALING PORTION of the REVERSE PRIMER. For example, we want the REVERSE PRIMER for pLtetO above. We want the primer to bind to nucleotides at the 3' end of the template (for example, atcagcaggacgcactgacc). Remember that PCR extension happens at the 3' end of your primer, so you want to REVERSE COMPLEMENT the portion of the template that you want the primer to bind to. There are many tools to do reverse complement, including http://www.bioinformatics.org/sms/rev_comp.html. For example, for the case of pLtetO, the REVERSE COMPLEMENT IS ggtcagtgcgtcctgctgat.
2. RESTRICTION SITE
  1. Same as in Part 2. For the pLtetO example, let's choose PstI (CTGCAG)
3. BUFFER NUCLEOTIDES
  1. Same as in Part 2. For the pLtetO example, PstI has 98% cutting efficiency with 3 BUFFER NUCLEOTIDES, 50% with 2 BUFFER NUCLEOTIDES, and 37% with 1 BUFFER NUCLEOTIDE. Let's choose 3 (e.g., GTA).
4. ASSEMBLE AND NAME THE SEQUENCE
  1. Remember that you are using the reverse complement of the template for your TEMPLATE ANNEALING PORTION.
  2. For example, for PstI-pLtetO-r we have 5' GTA CTGCAG ggtcagtgcgtcctgctgat 3' TmNN 66/56
Check your primers to make sure there are no self-dimerization or hetero-dimerization problems
1. Convenient tool at http://www.idtdna.com/analyzer/Applications/OligoAnalyzer/
2. In general you should look for PCR primers that conform to the following guidelines.
  1. The difference between melting temperatures of the primers should be less than 5 degrees.
  2. The GC content should be between 35-80% or equivalent to the product being amplified.
  3. Using the IDT software, you will want the Delta G value of both the self-dimer, hairpin, and heterodimer to be more positive than --9.0 kcal/mole. Positive numbers indicate the actual secondary structure pictured will not form at all. You will notice that you will get self-dimerization due to the fact that the restriction enzyme sequences are palindromes. For example, PstI-pLtetO-r has a maximum delta G (of the entire primer) of -55.82 kcal/mole and a delta G due to self-dimerization at the PstI site of -10.24 kcal/mole (see below). I try to keep the most negative delta G (in this case -10.24 kcal/mole) to at least 1/4 or 1/5 of the maximum delta G if I can.
  4. Avoid 3' dimerization or else you might get primer dimers. For example, the following example is very bad:
  5. How to fix this? We could add an addition few base pairs to the 3' end of the primer. For example, add AC. This doesn't change the fact that you have self-dimerization with a highly negative delta G (which is bad), but will at least help reduce primer dimers because the DNA polymerase will not be able to extend off the 3' end of the primer.

Design your PCR reaction

There are many different types of polymerase. We like Phusion because it has high speed and fidelity (http://www.neb.com/nebecomm/products/productf-530.asp).
For Phusion, usually I like to run 5 cycles with primer annealing temperatures around or higher (+2-3C) than the lower annealing temperature of the two primers (TmNN of the TEMPLATE ANNEALING PORTION). For example, for MluI-pLtetO-f and PstI-pLtetO-r, we have TEMPLATE ANNEALING PORTION TmNN's of 56C and 56C, respectively. Thus, I could choose to run the 5 cycles at 58C primer annealing temperature.
Then, I like to run 30 cycles with primer annealing temperatures around or higher than the total TmNN of the two primers. For example, for MluI-pLtetO-f and PstI-pLtetO-r, we have total TmNN's of 65C and 66C, respectively. Thus, I could choose to run the 30 remaining cycles at about 67C primer annealing.
For Phusion, they recommend 15 seconds extension time per 1 kb of low complexity DNA (e.g., plasmid) and 30 seconds extension time per 1 kb for high complexity DNA (e.g., genomic).
If there are issues getting product, try increasing extension time, reducing the primer annealing temperatures, changing buffers, etc.
RECIPE FOR PHUSION PREMIX (1.1X)
IDEALLY, prepare the PCR reaction mixture on ICE and also add the Phusion Premix LAST, since Phusion DNA polymerase has 3' -> 5' exonuclease activity and can degrade primers in the absence of dNTPs (although the Phusion Premix has dNTPs, so this is not as big of a problem).

Sample PCR Reaction Mixture

Name of Component	uL	Concentration
Template	Usually 1-2 uL	General guidelines are: 1 pg - 10 ng per 50 µl reaction with low complexity DNA (e.g. plasmid, lambda or BAC DNA); 50-250 ng per 50 µl reaction with high complexity genomic DNA. If cDNA synthesis reaction mixture is used as a source of template, the volume of the template should not exceed 10 % of the final PCR reaction volume.General guidelines are: 1 pg - 10 ng / 50 µl reaction with low complexity DNA (e.g. plasmid, lambda or BAC DNA); 50-250 ng/50 µl reaction with high complexity genomic DNA. If cDNA synthesis reaction mixture is used as a source of template, the volume of the template should not exceed 10 % of the final PCR reaction volume.
Forward Primer	1.25 uL from 20 uM stock	Final concentration 0.5 uM
Reverse Primer	1.25 uL from 20 uM stock	Final concentration 0.5 uM
Phusion Premix (1.1x Concentration)	Fill up to 50 uL total volume
DMSO (optional for troubleshooting)
TOTAL	50 uL

Sample PCR protocol - YOU CAN USE THE ATTACHED WORD DOCUMENT AS A TEMPLATE

Step Number	Cycle Step	Temperature	Time
1	Initial Denaturation	98C	30 seconds
2	Denaturation	98C	10 seconds
3	Primer Annealing	+2-3C above lower TmNN of the two TEMPLATE ANNEALING PORTIONS	30 seconds
4	Extension	72C	15-30 sec/1 kb	GOTO STEP 2 FOR A TOTAL OF 5 CYCLES
5	Denaturation	98C	10 seconds
6	Primer Annealing	+2-3C above total TmNN of the two primers	30 seconds
7	Extension	72C	15-30 sec/1 kb	GOTO STEP 5 FOR A TOTAL OF 30 CYCLES
8	Final Extension	72C	6 minutes
9	Finish and Hold	4C	Forever

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Copy of Primer Design Guidelines for PCR Cloning