Flanking Sequence Addition

Utilise your skills in PCR and Primer Design to ‘lift out a gene’ from a plasmid, genomic or environmental sample. Add restriction sites, or even the sequences for Gibson or Golden Gate assembly.
Use your Restriction Digest and Ligation skills to insert into a plasmid of choice.
This protocol can be tricky to pull off, but is insanely versatile.

Primer Design

All of the general Primer Design considerations apply for the addition of flanking restriction sites, however the one we’re hoping to make use of most is:

3’ Homology is much more important than 5’ Homology.

Thanks to this wonderful loophole, as long as 15-20 bases at the 3’ end of the primer are absolutely conserved from the target sequences, we can add up to ~30 bases to the 5’ end without negatively impacting the reaction. Since the primer is incorporated into the newly assembled strand, all of the subsidiary strands in the chain reaction will include your new flanking region.

These extra bases come with the associated risk of dimers and hairpins, so be sure to check that the ΔG is high enough, ie. > -5 kcal/mol

Design Considerations for Restriction Enzyme Digest:

Design considerations for Gibson Assembly:

Design consideration for Golden Gate Assembly:

Does your sample need pre-purification?

If you’re attempting to extract a gene from a natural sample, you may wish to use one of the following purification protocols. If you’re amplifying a gene already in the lab, you’ve probably already purified your target, so I won’t repeat this step here.

  1. Purification of Genomic DNA

  2. Purification of Plasmid DNA (Miniprep aka. Ethanol Lysis)

That said, Taq polymerase is a very robust enzyme. You may just want to drop in 1 µl of unpurified sample to see what you get.

Equipment and Consumables:

Set-Up Protocol:

  1. While you don’t technically need more than one PCR tube for this reaction, you may want to consider setting up a master mix for 4 tubes if your pipetting tolerance cannot handle these smaller volumes. Set up the following reaction on ice:

    • REACTION MIX (Final Conc.)

      • 20 µl Sterile dH2O

      • 2.5 µl 10x buffer (1 x)

      • 0.5 µl 10 mM dNTPs (200 mM)  

      • 0.25 µl 50 µM Primer #1  (0.5 µM)

      • 0.25 µl 50 µM Primer #2  (0.5 µM)

      • 0.25 µl (5 U/µl) DNA Polymerase (0.05 U / µl)

      • 1µl purified DNA template (plasmid or genomic)

  2. Aliquot out the master mix between all of the PCR tubes, putting 25 µl in each tube.

  3. Put lids on tubes, ensure they are snapped on tight, place immediately in thermocycler. Double check your program parameters before starting. See below for detailed thermocycling instructions.

  4. Return all reagents to the freezer.

Thermal Cycler Set-up

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Initial denaturation:  95°C, 5 min 
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Denaturation:          95°C, 30 sec, 25-35 cycles

Annealing:               X°C, 30 sec, 25-35 cycles

Extension:               72°C, Y min, 25-35 cycles
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Final extension:      72°C, 10 min

Hold:                       15°C      
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  • X = Annealing Temperature which primers will bind

  • Y = Extension Time that the polymerase will need in order to amplify your segment.

*You can leave the reaction to proceed overnight and start again in the morning*

Agarose Electrophoresis Analysis:

You should run an agarose gel to see if the reaction was successful and produced a consistently sized product. Since your primers are less homologous to the target sequence, it is common to see non-specific product in poorly optimised reactions.

Follow the troubleshooting tips on the PCR page if you’re failing to obtain consistently sized product, consider performing a gradient PCR to determine the perfect annealing temperature. Consider increasing the extension time if you’re obtaining lots of undersized product.

Purification

Based upon your agarose gel, you should know the quality of sample you’re working with. If you’re able to obtain a crisp tight band, you’re welcome to use any of the following purification protocols. However, if you’ve non-specific product bands close in size to your intended target, don’t use the column protocol, instead focus on using the gel extraction protocols, starting with the excision steps described on the gel electrophoresis page.

  1. Buffer QG Gel Purification (Industry standard but extremely toxic)

  2. Cotton Wool Gel Purification (Easy and safe, significant loss of yield)

  3. Buffer PB Column Purification (Skip the gel, just purify the PCR. Only perform if you’ve got a tight band on the gel and a spin column of the correct grade)

    • You can combine 4 PCR reactions into one tube to obtain the desired volume for this protocol (100 µl) or perform 2-4 separate purifications in order to balance your centrifuge.

If you want to be thorough, run another Agarose Gel to check that the purification was successful.

Downstream Reaction

You now have your desired gene, with fancy new flanking sequences. You could store this for a later date, however you should have had a downstream reaction in mind when you started this experiment. Proceed to one of the following protocols with your purified amplicon;

  1. Restriction Enzyme Digest

  2. Gibson Assembly

  3. Golden Gate Assembly