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Designing Gateway PCR Primers

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Biological and Medical Sciences > Classification by Description > Reagents, Kits and Assays > DNA and RNA > Cloning


Editor:  Christopher Dyer
Branch: Protocol-Wiki
Type: Protocol
Intention: For Publication
Read/Write Permissions: Open Access
Authors :
16.09.2010 - John Stewart   
13.09.2010 - Christopher Dyer  -  Sci-Mate   


A very easy way to insert a gene or sequence of DNA into a Gateway vector is by including Gateway ends on a PCR fragment, and Gateway cloning the fragment into a Donor vector via the basic Gateway cloning reaction. Once you have a sequence of DNA within any Gateway vector, it can then be very easily moved between vectors for expression, sequencing, tagging, and other sub-cloning purposes via the same basic Gateway cloning reaction.

Contents

[edit] Adding Gateway Restriction Sites to PCR Primers

Designing Gateway primers is essentially the same as designing normal primers, plus the inclusion of B1 and B2 restriction sites at the end of the forward and reverse primer sequence (see Gateway Lambda Phage Restriction Sites). This very simple Protocol assumes that users know how to design primers that will amplify the gene or sequence of interest in a way that ensures in-frame expression within any standard Gateway expression plasmid (for some tips, see Designing a Gateway Cloning Strategy). Once you have the necessary priming sequence:

The following sequence (B1 restriction site) is added to the forward primer: 

  5`-GGGG-ACA-AGT-TTG-TAC-AAA-AAA-GCA-GGC-TNN- rest of forward primer.

AND, the following sequence (B2 restriction site) to the reverse primer:

  5`-GGGG-ACC-ACT-TTG-TAC-AAG-AAA-GCT-GGG-TN- rest of reverse primer.

Where:

  • The 5`-GGGG end is there to protect the important part of the primier from degradation, and not part of the restriction site (nor are the Ns, see point below).
  • The bold underlined regions will remain with your sequence throughout all Gateway cloning reactions.
  • The two additional nucleotides at the 3` end of the forward primer are only required if there is a need to keep the product in frame with an N-terminal tag in the destination vector; and the single N in the reverse primer will keep the gene in frame with a possible C-terminal tag. Both can be handy options to keep open.
    Note: do not use AA, AG, or GA for the NN in the forward primer, as this would create a stop codon (the supplier recommends TC in most examples), and avoid stop codons all together in the reverse primer if you specifically intend to insert the gene into a vector with a C-terminal tag.
  • The inclusion of a Shine-Dalgarno and/or Kozak sequence into the forward primer to assist expression can assist expression in bacterial and/or mammalian cells (respectively). The following sequence contains the Gateway B1 and both Shine-Dalgarno and Kozak sequences, which is a handy option to ensure expression (note: an N terminal tag would be kept in frame with the bold Kozak ATG start codon in this sequence by the TC):

  5`-GGGG-ACA-AGT-TTG-TAC-AAA-AAA-GCA-GGC-TTC-GAA-GGA-GAT-AGA-ACC-ATG-G- rest of forward primer-3'

  • The inclusion of unique restriction sites is a way to bring in additional sequence via traditional cloning techniques.

[edit] Recommendations

Recommended actual PCR primer length is 18-25bps (forward and back).

When considering a source for the PCR product, avoid where possible using a vector confering Kanamyacin resistance (see Designing Gateway Cloning Strategy). Most Donor and Entry vectors contain this resistence gene, so if there is left over vector from the PCR, it will create background colonies in the basic Gateway cloning. If this cannot be avoided, quickly digest the PCR mixture with DpnI (just add buffer and enzyme; leave for 15 min at 37oC; and heat inactivate 65oC for 20 mins), which will cut all methylated GATC sites (which cannot exist within the PCR fragment).

[edit] PCR

There is nothing special about the PCR in a Gateway experiment.

Of course, use a proof-reading polymerase. Finnzymes polymerases are excellent.

A 50ul reaction is more than enough to run a few ul on a gel to check for amplification and correct size, and to then either use directly in Gateway ligation, or to purify, and then use in the ligation reaction.

[edit] Note

This basic Protocol is open for editing by exerienced users - more examples of sequences would be of help to other users. Discussion on the associated discussion page is of course welcome.





  • This page was last modified 19:08, 16 September 2010.
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