Dear MeghA

10kb is big but not impossibile. whixh taq did you used? i suggest to you to try

kapa hifi (roche)

clone amp (takara)

q5 (neb)

good luck

Manuele

Try using High-fidelity tag like Phusion from Thermo Scientific or long RA from Genscript ( you can use your 2.5 Kb products as template) then subclone all of them separately in pUC19 or any small size cloning vector.

Then you can do any type of assembly (ligation) of these 4*2.5 Kb products. You can use Gene builder kit from Genscript or you can add any one of three Type IIs enzymes (AarI, BsaI, and BbsI) and perform GeneArt Type IIs Assembly.

I had amplified an 8-kb sequence using XT-20 polymerase (GeNei). The enzyme is said to ampify upto 20-kb, so it should possibly work for the 10-kb sequence.

Hi Megha

I cloned the entire full-length dystrophin coding sequence (11kb) by PCR amplifying it using q5 polymerase. It worked very effectively. I also amplified a 16kb sequence from genomic dna using the same reagent and obtained a good yield, with high fidelity. It is described in this paper.

https://www.ncbi.nlm.nih.gov/m/pubmed/28250438/

If PCR amplifying long sequences, remember to reduce the number of PCR cycles. A long amplicon obviously has much greater mass than conventional PCR products, so you don’t generally need as many copies to see a band on a gel. Also, the polymerase can become exhausted when amplifying long targets, so it’s best to keep cycles to a minimum.

For the 11kb fragment, I used about 16-18 cycles. It’s also very important to try an annealing temperature gradient to minimise non-specific amplification.

Best

John

HI Megha,

Try Gibson assembly. This is very effective in cloning bigger segments. and As counsell suggested use q5 polymerase for PCR amplification.

Göran Hjälm

In terms of PCR fidelity, I did not observe any mutations. I loaded the sequence into a HIV-1-based lentivirus, transduced stem cells and then PCR-amplified the final product. Sanger Sequencing showed 100% fidelity, but it’s possible that deep sequencing could reveal a specific mutation rate across clones.