I am so disappointed that I failed in PCR again and again - (Sep/28/2006 )
I have designed 8 pairs of primers of HER-2 mRNA.
They were designed by DNAstar, Primer Expression, IDT, or copied from PrimerBank.
I have PCR with them time and time again, however, without success.
Only one pair successfully primed the right size product, but under extremely special condition: touch down PCR and 65 cycles; and another pair primed the target product size with a lot of primer dimers.
While, in normal PCR (35 cycles, 55 centigrade anealing temperature), the first pair produced a weak product band with some nonspecific bands in agarose gel, and the other did not.
The PCR program was as follows:
95 oC 5 min
94 oC 30 sec
Aneal Time *
72 oC 30 sec
72 oC 5 min
4 oC hold
The anealing temperature and lasting time were:
cycle 1: 66 oC 1 min;
cycle 2: 65 oC 1 min;
cycle 3: 64 oC 45 sec;
cycle 4: 63 oC 45 sec;
cycle 5: 62 oC 40 sec;
cycle 6: 61 oC 40 sec;
cycle 7: 60 oC 35 sec;
cycle 8: 59 oC 35 sec;
cycle 9: 58 oC 30 sec;
cycle 10: 57 oC 30 sec;
cycle 11~65: 56 oC 30 sec;
The product sizes vary from 110bp to 420bp.
I do not know why~
I am to use the primers on to real-time PCR cycler......
Why there is no result......
Holy burritos buddy. You sound ambitious, but try to break this problem down to something simple rather than diving into this all at once. First off are you doing RT-PCR or PCR are you amplifying from RNA or DNA. Secondly, this touchdown thing is not going to help. There is obviously a consistant problem you are having if ALL you rxn's are not working. Start with one reaction and try to work through the problems. Check your primer sequences, make sure they are on the correct strand/orientation. Check your template. Then check your reagents and reaction conditions.
I agree with tap14, go back and check your PCR technique first before trying to develop such specialized conditions. Here are a few ideas: I always chose primers with ~50% GC content and 63 degrees annealing temp works great for me. Try using a hot start taq, this can really help with the non-specific bands. Check the concentration of your primers. I use 0.2 uM final concentration in the PCR rxn of each primer.
You are truly ambitious
I have few question to ask:
1. are you running PCR or RT-PCR?
2. are you running singleplex or multiplex?
1. If you run multiplex, try to run singleplex first.
At least your singleplex must work first.
2. BLAST all you primers, make sure that they do not form primer dimer and hair pin loop among
3. Make sure your primers have similar Tm, so that they can hybridize at the same temperature.
4. Mind your extracton, quantitate and examine your extraction quality.
5. show us your gel photo.
Here are some common things I have found to trouble&shout ;-} standard PCRing.
1. Start with two or three batches of PCR buffer (One of the companies seminar I went to showed how Tris buffer pH are diffrent)
2. Start with dNTP from TaKaRa. I have found several sources of dNTP dissolved in water. Deamination of purine bases in acidic water would cause your PCR never working or working erratically.
3. Try three sources of Taq. Try TaKaRa Taq Polymerase as I have seen that it has consistently given excellent results.
4. Make sure you have MgCl2. Higher MgCl2 is safer than no MgCl2. One company I bought MgCl2 was only water...two weeks of frustration taught me this. It turned out nobody in the department was able to do PCR for two weeks. Issue was same batch of MgCl2..i.e. water. Never rule anything out.
5. Add upto 0.5% of Fatty-acid free BSA prepared in DEPC-water, or buy from NEB or TaKaRa.
6. Again addiing DMSO upto 5% may help against GC-rich sequences, with approx 5% higher Taq consumption.
7. Addition of basic amine, such as putrescine or betaine may resolve the strand separation in case of GC-rich PCR product.
Of course this involves a lot of investment, but you will love when things are sorted out and are proven to be worth spending little money to finish the project.
(I am not interested in helping TaKaRa, but it has the best product that helped me solve the problem making substantial time saving..of course TaKaRa can bring down the prices of their buffer and dNTP)