Someone who did gel purification for large DNA fragment(> about 8000bp) and took high DNA concentration is very welcome!!!

Your protocol looks like you didn't maintain the proportions of the vector and

your cut-out gel fragment. Look carefully at the protocol of Thermo

Scientific CloneJET PCR Cloning Kit and do same proportions. Or buy this

kit. It works great!

If you need to just ligate a small fragment of dsDNA into the linearized plasmid body (8600-bp piece), you do not have to purify the cut plasmid. It's sufficient to thermoinactivate the restriction enzyme and then perform the ligation using excessive amount of your dsDNA insert. Of course, you will get some side products, like with back-ligated cut-out piece or re-circulized plasmid without any inserts, but those can be eliminated later after transformation and colony screening.

If you still want to gel-purify the 8600-bp piece after cleavage, better look for electroelution to get it out of the gel. Dissolving the gel and capturing such a large fragment on a spin-column will surely be associated with huge DNA losses.

Irina Zyrianova

Thank you for your reply! I'm going to check that protocol!

Victor G Stepanov

Thank you for your reply!

I have two fragment of DNA which I want to ligate.

One of them is 8600bp and it comes from 11000bp plasmid.

Other one is 5500bp and it comes from 8600bp plasmid.

Can I ligate them with no electrophoresis and gel-pulification?

Or I will ask my professor today.

Well, if you aim to excise 5500 bp and 8600 bp pieces from separate plasmids and then ligate these pieces together, then it would be better to gel-purify them prior the ligation.

Actually, do you have any selectable marker on these two fragments, 5500 bp and 8600 bp? Antibiotic-resistance gene or something alike? And which restriction enzymes do you use to cut the original plasmids?

Victor G Stepanov

Thank you for reply.

I tried ligation with other plasmid which I use in my research, and it was success! This time, ligated plasmid is about 5000bp. From this, my professor and I think that failure on previous ligation was because of transformation after ligation of long read ligated plasmid. So I'm going to buy other E. coli strain which is suitable for long plasmid.

I have antibiotic gene and ori in one of fragment in that plasmid. And cut them with HindⅢ and XbaⅠ.

Ooi Yoshito Great that you could ligate them in the end! What was the ligation protocol that you used? I am also trying to ligate a 6,3kb vector with a 400bp insert but am still unsuccessful.

Umay Ece Tugcu Thank you for your reply!

I forgot to add later progress for my plasmid ligation which is mentioned at my first question. After that, I changed only competent cell strain for my protocol and I could take colonies. Result of colony PCR suggests that I may get optical plasmid. Of course, I will do sequencing later for my plasmid.

I used T4 ligase from TaKaRa and used their protocol(written in Japanese) and BioLabs protocol for T4 ligase. And I used E. coli HST08 from TaKaRa which is competent cell for long plasmid. How about use In-fusion kit for ligation ? It seems to be better at ligation efficiency than T4 ligase from my lab member's experiment.

I hope it can help you!