Dear all,

I have moved to the UK now, and joined Immunocore Ltd, close to Oxford.

I am trying to implement an high throughput cloning method, and I was wondering if any one of you would be willing to share some ideas / protocols.

I am trying to implement something like SLIC, or the RecA cloning method Sabine has been using with pCoofy vectors, but have a few questions on the scale-up of the throughput, namely:

- Do you run the PCR products on a gel (or capillary electrophoresis - this one is for Renaud!)?

- Do you need to quantify the PCR products (after purification, if that is needed)?

- Do you normalize the concentration of the inserts?

At this point I would be mostly interested in doing high-throughput "by hand", meaning - using multichannel pipettes...

Any thoughts on that?

Hope my new "Private Company" status won't stop any one of you to provide some help!

Thank you all!

Pedro

We are still using In-Fusion here with the pOPIN and pPEU vectors (but obviously can be used with any vectors) and for us the efficiency is high-normally we pick 2 clones and the vast majority of the time 1 or both are correct.

We run a sample on a gel (for In-Fusion gel purification is unnecessary) and there are many gel tanks out there now for quick sizing of fragments. Caliper etc. give very nice data (expected band present/absent, concentration, purity etc.) that might be useful if you are processing lots of samples and are using a cloning method sensitive to insert concentrations -but at a price. If we have the same antibiotic resistance in source and target vectors we DpnI treat and then use Ampure to purify the products, if resistance of template is different to target then Ampure only.

In-Fusion or one of its many variants now seems to be the method of choice for commercial cloners (I think Genscript and NZYtech for example use it) and there are also now many different sources of the enzyme under different names. Part of the appeal is that it can be very sensitive i.e. can pick up clones from very low PCR yields (sometimes in a plate processed 'en masse' we have clones from PCR products not visible on a gel) so normalization, which can be a pain, is unnecessary on the whole.

Doing this by hand with multi-channel pipettes, plate magnets and beads, plate shakers etc. is fairly simple, one person should easily be able to clone quite a few plates a month. Bear in mind that for every week of cloning you do you will have a couple of weeks of primer designing and sequence data analysis to do. It really depends on what you mean by HTP-there is HTP by most peoples' standards and then there is Renaud!!

And then, of course, there is the expression screening...