hello Jothivanan,

You could take in consideration couple of possibility, since 100bp is a length that all the major companies can synthesize easily:

1 (cheaper). Order a single 100bp oligos (from Sigma, IDT etc) and a couple of complementary primers (+/- 20bp) : then you can set up a high fidelity PCR ( i.e. NEB Q5) to fill up the double strand and amplify your template. You could also consider to use a single primer and just fill the double strand.

2. Order both 100bp oligos (rev and fw), incubate the together at 95°C for 5 mins and let them re-anneal slowly at RT.

Both this approach can be considered "artificial" and your final double strand amplicon won't have any epigenetic markers on, it beside a 3' phosphate (that can be easily eliminate).

Hope this might help

Fil

hello Jothivanan,

You could take in consideration couple of possibility, since 100bp is a length that all the major companies can synthesize easily:

1 (cheaper). Order a single 100bp oligos (from Sigma, IDT etc) and a couple of complementary primers (+/- 20bp) : then you can set up a high fidelity PCR ( i.e. NEB Q5) to fill up the double strand and amplify your template. You could also consider to use a single primer and just fill the double strand.

2. Order both 100bp oligos (rev and fw), incubate the together at 95°C for 5 mins and let them re-anneal slowly at RT.

Both this approach can be considered "artificial" and your final double strand amplicon won't have any epigenetic markers on, it beside a 3' phosphate (that can be easily eliminate).

Hope this might help

Fil

Hi Jothivanan,

With our local cores (Harvard/MGH), we have had significant issues with anything longer than about 55 nucleotides. After cloning PCR products, we inevitably have to pick the one with the longest correct sequence, then design a second oligo to add/correct the missing upstream sequence. This strategy works if the oligo is for PCR, but is much more difficult to accommodate if if one is trying to clone a ds fragment. Your experience may (hopefully) be much different with other synthesis sources though, as Filippo mentioned.

As I think you anticipate, if during synthesis one has, say, even a 99% accuracy rate at each addition step, the problem is that a greatly decreasing percentage of product is 100% correct in very large oligos.

Another useful company is MWG, http://www.eurofinsgenomics.eu/

This company and Sigma will happily synthesise over 100bp DNA molecules for you. Then you just need to decide whether to PCR the second strand of buy both an anneal as Filippo said.