Confirmation of Edits

There are many strategies available for screening recombinant clones. The simplest experiments involve a recombinant that has an obviously different phenotype to the unedited original – e.g. if you are cloning a resistance gene or a fluorescent protein. Just pick up the glowing colonies and you’re good to go. You may even have a more specific system such as the classic blue/white colony screen.

However, in most cases, we will need to screen the surviving colonies to find the truly successful recombinants that we are seeking. All kinds of funky things can go wrong with ligation, and simply getting a colony to grow on your ligation plate is no true indication of success. Undigested plasmid is the most common problem, but flipped genes, double inserts and even contamination from other experiments might explain that growth.

I generally do these protocols in order, just to be certain I save money - but the future will not require such manual work. Soon sequencing will be cheap enough for all of us to skip straight to Step 3 or 4 - but until then, rev up your thermal cyclers!

1) Patch Plate and Colony PCR (Junction & Spanning PCR)

A patch plate is a way to organise promising colonies for a PCR. You can skip the exhausting purification steps by performing a “Colony PCR” - adding whole cell mass to the PCR tube and relying on the denaturation step to lyse the cells for polymerase access. Junction PCR uses specific primers to create an amplicon spanning the ligated restriction sites of interest and is the most reliable. A spanning PCR creates an amplicon that covers the entire gene of interest, but comes with the chance of false positives.

2) Restriction Digest Analysis (+ Miniprep & Agarose Gel)

Once you’ve identified likely successfully ligated plasmids, you can now justify the expense and time for purification and restriction digest. Use software to choose an enzyme inside your inserted gene and inside the backbone, then run an agarose gel to see if the size of the bands match your modelling.

3) Preparation for External Sequencing

If you’re a baller and can swing the money for external sequencing, why not just make absolutely certain. Purify your plasmid or a spanning PCR and then send either off for sequencing by an external company.
This company will use either a) Sanger Sequencing or b) 2nd Generation sequencing to sequence your sample and will then send you the sequencing info either BAM or FASTQ file format. There are various software options available for interpreting this data.

4) In-House Sequencing

If you’re truly blessed with finances and connections, perhaps you’ve access to your own 2nd gen sequencer.
Otherwise, the Oxford Nanopore is the first device that is within the grasp of wealthier hobbyists and promises a future where anyone can gain access to cheap DIY sequencing. I will add protocols to this section once Oxford Nanopore finally sponsor me ;)