I get better luck with using a high-fidelity polymerase that leaves an A overhang and cloning into a TOPO plasmid vector. No need to fuss around with restriction digest, ligation, etc. The kit is expensive, but it works so well it's worth the money.

https://www.thermofisher.com/us/en/home/life-science/cloning/topo/topo-ta-cloning.html

Hi Sakil,

Have you ever done PCR with regular taq polymerase/PCR mix to check the expression levels of your isoforms in the cDNA you are using?

High fedility polymerases which can amplify 2kb easily are available commercially. In you case iProof high fidelity polymerase (since you amplified 2bands already) can be used with increased amplification time and cDNA made from freshly prepared RNA (try to be gentle and careful with RNA which is prone for degradation of longer sequences).

Second usage of less plasmid (>1ug) is preferable. Better change gel extraction kit. Ligation reaction for overnight at 16degrees is better for longer fragments. All the best.

Dear Sakil.

Two things spring to mind based on your answer but I think you might have multiple things going on:

1. You mentioned that you didnt get the longest product of your 3 isoforms from that Biological cDNA sample

2. Whilst this could simply reflect reaction kinetics; that is large products are amplified less efficiently than smaller products. That being so, as already suggested, you could try and optimise your reaction conditions for the longer product. Another thing you could try with your PCR apart from already suggseted is to

• Add an initial denaturation cycle of 95C for 5 min before commencing PCR proper
• Perform an annealing gradient with you your Taq/polymerase, where in multiple preliminary PCR reactions you vary the annealing temp in 1C intervals from Tm-5C to the actual TM, i.e. 5 x reactions @ Tm-4C Tm-4C ...etc.: Sometimes for larger products you might want to relax the stringency of your PCR reaction (without making it non specific) to encourage efficient PCR amplification of longer products in particular:
• To elaborate, generally a Tm-2C for your annealing temp is about right for most PCRs but for certain PCRs, particularly those involving products larger than 1.5kb, for all polymersases - including highly processive PCR polymersases, more suited to efficiecnt amplification of larger amplicons, more 'relaxed PCRs wherein the annealing temp is as low at Tm-5C can tip ther balance in favour of efficient amplification of larger specific amplicons without causing concomitant amplification of different sized non specific products (< Tm-5C will significantly increase the risk of non specific amplification without increasing specific priming and hence the amount of specific amplicon) simply based on reaction kinetics
• In Addition, I would also add 5% DMSO to your PCR reaction: That will help reduce any secondary structure that tends to be more prevalent with larger products

@ CHANDRA SHEKHAR DASARI

Yes, I have used regular Taq polymerase also but there I could not find any band? But in case of high fidelity polymerase I got the bands.

Dear Laurance

Thanks for your suggestions. Answers for your asking is folloeing:

1. Do you have eny evidence that the longer isoform is expressed in your tissue ?

Yes, one of the isoform I found in the article they are located in the cytoplasm. Actually, I am checking the localization of these isoforms in cell compartments.

2. Yes, I also tried with actin and I got the band from my cDNA also.

I have another qurey to you, Could you please tell me any special feature of pBIN19 if you can?

Regards

Hi Chandra

Thanks for your reply

Yes, I have used Taq polymerase also. But, there I could find any band.

And I also run a temp. gradient also by i-proof and there I got the bands for two isoforms clearly.

Sorry Sakil

dont know about pBIN 19 vector