If your friends get a pcr product from the same dna then either the primers you use are not working.......degraded or Ta is too high) or their bisulphite treatment is working better. Try borrowing some dna from them and run an assay with both their dna and primers and their dna and your primers to sort out where the problem is. Can you post the primer sequences and the annealing temperature that you are using for each amplification please?

This is the primers I uses. For the annealing temperature actually I have already tried 50-60 degree celcius for both each nested and MSP for these 2 gene. But before changing into this mastermix 53 degree celcius for nested and 56 degree celcius for msp works best for InsR. As for slc2a4 currently I'm still trying optimize it again.

my feeling is that it is unfortunate to have a long (600bp) amplimer using low temperature annealing primers because the template dna at low temperatures will

re anneal quicker than the primers can anneal so you have a low efficiency pcr. Ideally longer primers or a shorter outer sequence at first amplification could work better . Still these are the primers that you have so you could try running the pcr in presence of 1M betaine final concentration to try to stop the template reannealing for as long as possible or try adding 6%dmso to the pcr mix to stop secondary structure formation again to hold the strands open for as long as possible . For slc i would run a gradient from 45 to 55 annealing for the first amplification even if this only provides a dirty amplification because it is hard to troubleshoot a complete failure but relatively easy to clean up a dirty amplification

And also do you know what caused this? This is InsR gene

the outer primers seem to be amplifying a band of about 500bp as expected but also some non specific amplimers. If this picture is just the amplification with the outer primers then I would accept that the first amplification is usually poor which is why people use nested primers to clean up the dirty product

This is the second amplification, is there anything i can do to fix this dirty amplification?

How much first round amplimer are you using for the second amplification? How many cycles of pcr are you running for the second amplification. What are the second pcr conditions (temp and time).. Do you clean up the first pcr before running the second pcr or just use a dilution of the whole pcr mix Stanley Christy Satriya Jati Karundeng

Each round i'm using 3 microlit of amplimers, 30 cycles. AS for the time and temperature : 94C - 30 min, 53C : 1min, and 72C - 1min, and yes I'm uing dilution of whole pcr mix

I think that the problem is that you are using too much first round product and too many pcr cycles.

Primers are in huge excess in pcr so taking 3ul of product is transferring a large amount of outer primer to the second reaction so your pcr band is a mixture of outerf-outer R, outerF-innerR and innerF-outer R as well as innerF-innerR.

Also 30 cycles is huge amplification. 10 cycles is 1000 times more product and 20 cycles is 1000000 times more product which is huge over amplification.

I would take 1ul of first round product (unpurified) and dilute it to 100ul.

Then set up 8 tubes of pcr each with 1ul of the 1/100 diluted pcr product and set the pcr machine for 26 cycles. At cycle 12 remove one tube then at 14cycles remove the next tube and remove another tube every 2 cycles. Some of these tubes should give strong clean amplification and the higher cycle numbers will probably be messy and over amplified but it will tell you how much template and how many cycles for the nested pcr