What is the od260/280 ratio of your dna sample. I expect the fluorescence in the well is protein and that the pcr is not working because the dna is impure. There is a lot of material in the well and this may be poisoning the pcr by removing Mg from the pcr reaction mix. There is also a lot of primer showing on the gel and too much primer can also remove mg and stop the pcr. 5.6 kb is quite a long amplification and is likely to have secondary structure issues so if your enzyme comes with a high GC buffer then use tis for the amplification but I think that the most likely cause of the failed pcr is impure dna template

Also do not forget that only the part of the primer specific to your GOI can be used to calculate annealing temperatures. The restriction site and extra bases do not bind to your dna for the first few cycles of pcr

Thanks for your prompt reply Paul Rutland . The A260/280 was 1.89 and A260/230 was 2.5. Earlier I did get amplification with primers having only restriction sites (Ta being 67.5) but I am not getting amplification after the addition of extra bases to the primer sequence.

What enzymes are you cutting with please?. Not all enzymes need the extra bases to cut satisfactorily.Often a RE will cut 75 or 50% efficiently without the extra bases so may just need cutting for longer Have you tried amplification with the short primers then dilute the product 1/1000 and reamplify with the longer primers for about 20 cycles at the short primer annealing temperature. It may be that the random extra bases at the 5'end of the primers is allowing a primer dimer to form spoiling the PCR in which case adding up to 10% dmso might improve the pcr with the longer primers. A gradient of dmso from 0-8% on one sample that works would be interesting....say 0, 2%, 4%, 6% and 8% dmso

I am using the enzymes XbaI and EcoRI. These enzymes perform well with additional bases, as mentioned on the NEB website. The incubation at 37ºC was done for 3hrs with XbaI first, and the heat inactivation at 65ºC for 20min followed by digestion with EcoRI for 3hrs.

Regarding amplification with the short primers, I did try, but using a 1/10 dilution and 35 cycles. The results were still negative but I shall give it a try again as per your suggestion. The gradient with DMSO will surely be performed since I had used 3% DMSO earlier. Thanks Paul Rutland for your suggestions.

I still have a query: why are bands near well seen in some PCR products while missing in others, despite all having the same PCR mix and a difference of only Ta? I am attaching the gel profiles for your perusal. The second gel profile has genomic DNA in the second last lane. The DNA alone has migrated more while the PCR products are near the well. What might be the reason for such a difference in banding patterns near the well?

Hi Nitesh Prasad . You have to be careful with re amplifications...10 cycles is 1000 times more product so 30 cycles is 10exp9 more product which is far too much and the result can be a long smear of amplimer to amplimer binding and extension and I think that your band close to the well could just be long amplimers running out of the separation range of the gel.

It is a shame that NEB do not publish the 'no extra base' result. As you have a pcr product with the short primer pair it would be interesting to amplify 1 1/1000 dilution of this product with all of the primer combinations....shortf to shortr, short f to long r, long f to short r and long f to longr as an aid to identifying which of the long primers is a problem.

The only dna sample in gel 2 suggests that you are using too much dna.....30ng is fine for 50ul reactions but may be too much for smaller volumes so it may be worth running a sample at dilutions of 1, 1/2, 1/4 and 1/8 to see if there are pcr inhibitors present which may be affecting the reaction

Thanks Paul Rutland . I shall get back to you after performing the task. Once again, thanks a lot for your suggestions