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Please help! no PCR bands+Description of my PCR protocol provided :( - (Jun/10/2011 )

Hey Everyone :D

I am doing my research project and I am having problems lately with my PCR. Please if anyone can tell me the problem I would appreciate any feedback. I ran a PCR two times and was unsuccessful.
I ran a gel of my PCRs but got no bands two times.

My PCR protocol is as follows:
Volumes as per PCR tube
50mM MgCl2: 2ul
50mM 10X PCR Buffer: 2ul
dNTP: 0.75ul
Forward Primer: 0.75ul
Reverse Primer: 0.75ul
Taq Polymerase: 0.2ul
cDNA template: 1ul (concentration is 40ng/ul)
DEPC-treated H2O: add up to 20ul

PCR STEPS in PCR Machine:
Step1: 94C for 3min
Step2: 94C for 45sec
Step3 (annealing): 63.2C for 30sec
Step4: 72C for 1min30sec
Repeat steps 2-4, 33 times
Step5: 72C for 10min
Step6: 4C for Ever
NOTE: my primers Tm ranges in the 68-72C range

I know this is a lot of information but I wanted to give as much detail as possible to make it clear. Please if you have any questions regarding my method feel free to ask. Any hints that can help me gain results would be great so post them!
Thanks :D:D

-yasamino-

have you done control PCR (actin or some known working primers) in the same cDNA?, doing so you can rule out there is no prob with the cDNA, then never stick to the same annealing temperature, the melting temperature of your primer will differ in the reaction mix so it is advisable to do a gradient pcr once you didnt get the amplication in a single reaction. i would suggest you to increase your annealing temperature further more (minimum 65) for your range of tm. Finally check the GC content of your region of interest if it seems to be more than 70 percent you may have to add some adjuvants like DMSO.

Gnana...

-GNANA-

could we have the sequence of your primers?

The annealing temperature is rather high.

Also what is the expected PCR product size?

-perneseblue-

GNANA on Sat Jun 11 12:17:19 2011 said:


have you done control PCR (actin or some known working primers) in the same cDNA?, doing so you can rule out there is no prob with the cDNA, then never stick to the same annealing temperature, the melting temperature of your primer will differ in the reaction mix so it is advisable to do a gradient pcr once you didnt get the amplication in a single reaction. i would suggest you to increase your annealing temperature further more (minimum 65) for your range of tm. Finally check the GC content of your region of interest if it seems to be more than 70 percent you may have to add some adjuvants like DMSO.

Gnana...


Hello Gnana,
Thanks for your feedback.
I have not done a control PCR. But I will do that for sure!
I will also do a temperature gradient for my PCR to know what annealing temp. is best.
Those are great ideas!
Thanks sooo much :D

-yasamino-

perneseblue on Sat Jun 11 19:59:12 2011 said:


could we have the sequence of your primers?

The annealing temperature is rather high.

Also what is the expected PCR product size?


HELLO perneseblue:D:D

Thanks for your reply as well.
The sequence for my primers is as follows:
I work on two genes, UB1 and UB2 (ubiquitin conjugating enzyme) in tomato.
1.
Sequence ID: 388206
1 ctcttcttcc atttctttca aaattaaagt attgttactc tgctattggc tcaaaacctc
61 tgcaatctcc gtctccttca atttcaactc aagcaaatcc acctctttca ctagtttcat
121 cactttcaga tcagggtttg gagttgaagg tacggggggc taattgatgg cgtcgaagag
181 gatattgaag gagctcaagg atctgcagaa ggatcccccc acatcatgca gtgctggtcc
241 agtggcagag gatatgttcc attggcaagc aacaatcatg gggcctaccg atagccctta
301 tgctggaggt gtatttttgg tttcaattca tttccctcca gattatcctt ttaagcctcc
361 aaaggttgcc ttcagaacta aggttttcca tcccaacatc aacagcaatg gaagtatttg
421 tctggatatt cttaaggagc agtggagtcc agcattaacc atatccaagg tcctgctgtc
481 catctgctct ctgttgacag acccaaaccc agatgatcct cttgtacctg aaattgctca
541 catgtacaag actgacaggg ccaaatacga aaccactgct cgtagctgga ctcagaaata
601 tgcaatggga tgatgcgcaa aatgtctcca ggcatgtctg ggactttgta acagcaatgt
661 cttatgtgct tggggtgaat gaataaattc cgtgaaagaa cttagttact tcttaatctc
721 ccttcatgag ggttgttaag ggaacagctg ttttcaattt gtgaatattt atttgatgac
781 tagtaaggga gaaactgcaa tgtaattcta ctttgtttgc cagtt
Primers

Forward Primer (NdeI)
5 c a t a t g a t g g c g t c g a a g a g g a t a t t 3

Reverse Primer (XhoI)
5 c t c g a g t c a t c c c a t t g c a t a t t t c t g 3

2.
Sequence ID: 886678

1 gcacgagggc gacttttgca taaaccaaaa ttagaatcaa attggaagag agaaaaaaaa
61 tggtggactt ggctagggtt caaaaggagc tccatgaatg caacagagat gttcaggttt
121 ctggaattaa tgttaccctt aaaggtgaca gtctcactca cttgattggt acaatccctg
181 gtcctgttgg tactccttac gaaggcggta ctttcaagat cgatatcact cttactgatg
241 gctacccatt tgagcctcca aaaatgaaat tcgccacaaa agtttggcat cccaacataa
301 gtagtcaaag tggagcaata tgcctagaca tcctgaagga ccagtggagc ccagcactaa
361 ctctcaagac agctctcctt tctatacaag cattactttc tgctcctgaa cctgatgatc
421 cacaagatgc agttgttgca cagcagtatc ttagagaaca tcagaccttt gtcggcacag
481 ctcgttactg gactgagact tttgcaaaaa catccacact tgctgcagac gacaagatac
541 aaaagcttgt ggaaatgggc tttcctgaag ctcaagtgag gagtactttg gaagcaaatg
601 gttgggatga aaacatggct cttgaaaagc tgttgtccag ctaaaaccct tctactgcaa
661 ctcatatttt gataagacaa ttatatcctt ccagcaaaag ctgatgacta gaatagagtc
721 actcggttat actgttgctt ggcaatcttg tttctgtctc ctttatggtt tgctgttgac
781 atctcttcat atcctgtgaa gattctgatg ttatttttac aatatcaagc aaattgcata
841 tgaatcatgg ggaggaagtg gactttccgg ggtgaaaaaa aaaaaaaaaa aaaaaaaaaa
901 aaaaaaaaaa aaaaaaaaaa aaa

Primers
Forward Primer (NdeI)
5 c a t a t g a t g g t g g a c t t g g c t a g g g t 3
Reverse Primer (XhoI)
5 c t c g a g t t a g c t g g a c a a c a g c t t t t c a 3

In terms of annealing temp. I thought it would be an ok one, but I guess I will try to do a temp. gradient to figure out the best one possibly.

My PCR product sizes are:
sequence ID: 388206 product length is around 447
sequence ID: 886678 product length is around 585

Hope that is clear :D

Its hard to decide what to manipulate in the protocol.

Thanks again :D

-yasamino-

Analysing your primers it doesnt seem to be of the tm range you have mentioned, it is far lesser than that...to my prediction it is around 55 degrees only (Excluding the overhang RE sites)...so i think your PCR should work fine if you set your Annealing accordingly, may be i would set between 53 and 55 as annealing for this tm...

Good luck...

-GNANA-

Your primers have RE sites at the 5' end, but are missing the required 5' overhang needed to allow you to cut your final product. This should not affect your PCR reaction, so you can optimize it using your existing primers, but you probably should order new primers, which will be needed for your next step. I'd recommend at least 4 bp of 5' overhang.

-phage434-

GNANA on Sun Jun 12 07:43:59 2011 said:


Analysing your primers it doesnt seem to be of the tm range you have mentioned, it is far lesser than that...to my prediction it is around 55 degrees only (Excluding the overhang RE sites)...so i think your PCR should work fine if you set your Annealing accordingly, may be i would set between 53 and 55 as annealing for this tm...

Good luck...


Thanks Gnana for your feedback. When I ordered my pimers, the Tm written on their tubes was in that range :$. But I will definately try that annealing range you mentioned.
:D

-yasamino-

phage434 on Sun Jun 12 15:27:52 2011 said:


Your primers have RE sites at the 5' end, but are missing the required 5' overhang needed to allow you to cut your final product. This should not affect your PCR reaction, so you can optimize it using your existing primers, but you probably should order new primers, which will be needed for your next step. I'd recommend at least 4 bp of 5' overhang.


Hello Phage 434
Yea I will look into that. I believe you are right. My professor also talked about this issue and hopefully will be solved.
Thanks Phage :D:D

-yasamino-