Sauer:Stitching Genes by PCR

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(New page: ==Introduction== This method allows you to "stitch" genes or coding sequences together when there are no convenient restriction sites at the junction point. It is espcially useful when th...)
(Protocol)
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*Step 3:
*Step 3:
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PCR your appendage.  If it is small (like a His tag), you can just have the two primers form a cassette when annealed.  In this case, the appendage is a bit bigger, so PCR is used to make the product from another template that contains the appendage.  Only do about 20 cycles, you don't need a lot and this will reduce errors.
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*Step 4:
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Set up a PCR with a microliter of the "appendage" PCR reaction in step 3 and a microliter of a miniprep containing your plasmid with your gene of interest.  Use primers "stitch forward" and "B reverse".  You will end up with the product shown.  I usually gel purify this product for the next step.  Again, you don't need a lot, use about 20 cycles.
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*Step 5:
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Set up a primer extention reaction with the gel purified product from step 4 and your plasmid.  Use about half of the gel-purified product from step 4.  Set the extension time long enough so the polymerase will extend to the left restriction stite "A".  Both strands of the PCR product will act as primers and you will end up with a heterogeneous population of single-stranded products.  The products that you need are the "bottom strand" extensions that primed back toward your site "A".

Revision as of 17:36, 10 March 2007

Introduction

This method allows you to "stitch" genes or coding sequences together when there are no convenient restriction sites at the junction point. It is espcially useful when the target gene is flanked by other genes that can't be disrupted. The technique is by no means new, but this should keep people from asking me how to do it.

Protocol

Refer to this figure:


  • Step 1:

Scan your sequence and find restriction sites anywhere to the left and right of the gene (the left one can be in your gene). Make sure they are unique and they are not contained in the fragment you want to append to your gene. In the figure, they are marked "A" and "B".

  • Step 2:

Design 4 primers. "A forward" primes toward your gene and anneals to the left of (or on) the restriction site "A"; "B reverse" primes back toward your gene and anneals past (or on) restriction site "B"; "stitch forward" anneals to amplify your appendage and has a tail that matches (anneals to) the left side of the fusion point (green in this case); 'stitch reverse" reverse primes your appendage and has a tail that encodes the region just to the right of the fusion point. The dashed lines show where the primers match.

  • Step 3:

PCR your appendage. If it is small (like a His tag), you can just have the two primers form a cassette when annealed. In this case, the appendage is a bit bigger, so PCR is used to make the product from another template that contains the appendage. Only do about 20 cycles, you don't need a lot and this will reduce errors.

  • Step 4:

Set up a PCR with a microliter of the "appendage" PCR reaction in step 3 and a microliter of a miniprep containing your plasmid with your gene of interest. Use primers "stitch forward" and "B reverse". You will end up with the product shown. I usually gel purify this product for the next step. Again, you don't need a lot, use about 20 cycles.

  • Step 5:

Set up a primer extention reaction with the gel purified product from step 4 and your plasmid. Use about half of the gel-purified product from step 4. Set the extension time long enough so the polymerase will extend to the left restriction stite "A". Both strands of the PCR product will act as primers and you will end up with a heterogeneous population of single-stranded products. The products that you need are the "bottom strand" extensions that primed back toward your site "A".

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