FYI !

Thanks everyone for your suggestions and help.

I had set up pcr with new working stock of primers. And all the bands got amplified. I even changed the positive control sample and it worked.

The problem seen in my pcr was primers which had got degraded due to freez thaw cycles.

please check the attached image.

Here too some samples have worked and others not.

Have you tried reducing the concentration of template? It worked for me once.

Hi

Try varying the annealing temperature and also use freshly diluted primers.

From your first picture it seems that there is some kind of contamination as the lanes are smeary. Make reagents at one place and set up PCR at other place. Be sure to include negative control(contaminants can make PCR worse)

Cheers.

From your first gel, it almost looks like there is actual genomic DNA in the lanes (evidenced by the bands just under the wells). To much DNA can "gum" up your reaction and cause the issues you are having. I would quantify your tail DNA samples and then make a working stock of template by diluting to 50ng/uL and use 1uL per reaction, 35 cycles in a standard PCR cycling profile.

If this doesn't work, please re-post and we can discuss further steps.

All the best.

Barry

There are strong primer dimers in your gel too. What are your PCR conditions and your primer sequences? Agree with others, there is DNA there and maybe you have too much ... another reason could be your primers are just annealing or self annealing preventing amplification. Denaturing your primers 95C 5 mins then put on ice may help, Q solution, increasing annealing temp....all this may help in addition to diluting your DNA. Set up a gradient temp PCR and vary all the above inc mgcl2 and buffer ph. Some companies sell kits that help you with this.

Good luck

Probably use a bit higher concentration of primers. Try also using new polymerase for PCR. Cross verify the extraction steps. I think, the PCR block might have problem since few samples showed better band and the others not. This might lead to disturbances in the annealing temperature and no amplification. May be this is also the reason that you see smear on your gel but no PCR product. Try with a new PCR machine. May be it works.

Good Luck

There could be several reason as mentioned above. But, two things are clear as mentioned above: high concentration of template and primer dimer. I did not understand what positive control. Is it for template like different set of primer and same genomic DNA or is it for primers like different batch of genomic DNA or plasmid for the same set of primers to see that is working or not. I will suggest to first use some other batch of DNA or PCR product or plasmid which have binding site for this primers to check wether these primers really work or not. Did you ever get these primer working before or this is first time these primer. If your primer had worked before then re-purify your genomic DNA or look primer is degraded or not. Trying gradient PCR with varying condition is best to optimized condition. In my case some time PCR machine cause problem. It was like my primer optimised for MJ Research machine cause non specific product or smear on other company PCR machine. So you have to go back to original machine where you optimised or re-optimised it for new machine.

Thanks all for your suggestions.

Well here the positive control sample is the genomic DNA sample which had worked earlier.

well the new set of primers had worked and gave good amplification earlier. It is the same set of primers which I am using it.

I will set up a pcr again with all your above mentioned suggestions and update my gel picture again.

profile:

Initial Denaturation: 94degree - 3mins

Denaturation : 94 degree- 30secs

Annealing : 65 degree-1 min

Extension: 2min

final extension : 7 mins

If i understood your reply correctly: your positive control with the same set of primers with old genomic DNA, that had worked earlier, gave amplification this time. But, with the same primer and condition and same PCR machine, your new DNA is not giving any amplification.

If I am correct, then your second batch of genomic DNA is problem:either it contains impurity or it is high concentration in reaction mixture.

I think you do again phenol chloroform extraction and then quantify along with old genomic DNA of your positive control on the same gel or by same method.

Then do the PCR.

Hope it will work.

Yes I meant the same as what you understood.

Thanks Mr.Parmit singh.

FYI !

Thanks everyone for your suggestions and help.

I had set up pcr with new working stock of primers. And all the bands got amplified. I even changed the positive control sample and it worked.

The problem seen in my pcr was primers which had got degraded due to freez thaw cycles.

please check the attached image.

G8 news Megha. Enjoy

I would suspect your DNA quality. Try a reprecipitation and carry out PCR and it may work. Or else start with a new batch of DNA. You also can check your earlier DNA sample that worked along with your fresh batch to make sure that PCR is alright

I am having a similar issue, I am getting very weak bands, almost unnoticeable, but my positive control is working fine. I've used this sample before and it has worked perfectly and I have tried other peoples aliquots of the same sample which have also failed.

Any suggestions?

Thanks

Hi Alan, please try using fresh template with new aliquotes of primers which would bind to gene. when you see the band of interest, gel elute it and extract the dna from the gel. Than, reamplify it.

So my question is, if your positives work but your DNA doesn't show up on the gel - changing the primers might help? If the primers degraded, wouldn't the positive control also not show up?

Dear all, 

I writing here as I think I can receive clarifications about troubles with my PCR amplification. I have to amplify a gene of 4000bp, so I designed my own primers for the 5' and 3' as I need to introduce RE sequences for cloning. these primers are new. I am extracting the RNA from several cell line, THP-I and HepG2, and from the last cell line I extracted proteins and made a western blotting that showed me the presence of my protein.

The problem is that after Reverse transcription of total RNA and amplification with the specific primers I am not able to get any band into the gel. I changed the RT conditions (random primers, and oligo dt), and used different kit and enzymes for the PCR amplification, changing temperature of annealing, adding Mg2+,  but nothing unfortunately.

Could you give me a suggestions about the possible problem?

PS: I am attaching the last gel (you have 4 cell line, and for each 3 wells, 2 samples and 1 beta actin that is visible).

Thanks

Gianmichele

I would suspect your cDNA      synthesis first. You have to make sure that your RNA prep was fine and the cDNA was OK. To make sure I would advise you to design two sets of primers of smaller length to make sure that you have the complete RNA corresponding to 4kb.What about introns?You need to thinks of all these and then design your experiment to get the desired result.

The RNA extraction was fine with a good concentration and purity. I checked the quality of my cDNA by using two primers for beta actin, that was amplified as showed in the figure. My cDNA synthesis by reverse transcription could be an hypothesis as it is 4000bp, so quite long, but not the longest. I used two different system, random primers and oligo dt without to geg any results. Should I move on the specific primer? I used in the past also a set of two diagnostic primers, covering a length of 200bp  at the 5'  end but nothing as well. What is your idea about introns? 

Do your primers correspond to the exon region or intron region of the gene that you wish to amplify. Check this point. It the primers are OK I would advise to get another batch of primers as these primers sometime may go bad.

Of course primers have been designed for the exon sequence absolutely. I designed primers on the basis of my mRNA, that should be done by exons only. 

I used already just one set of diagnostic primers at the 5' end, covering a distance of 200bp, but nothing as well. Honestly I have not idea what's happening. The beta actin primers on the same cDNA worked....but for my gene no results. 

I can only suggest another set of RNA to start with for cDNA synthesis or try a different primer combination

Megha Achar... but if the primers where the problem, then why positive control still amplified?

Absence of the sequence(s) of the primer(s) within the genome/gene means no hybridization. Consider: Your positive control has the hybridization sequences for your primers and yields the expected PCR amplicon(s). You need to walk the primers away from that location, one after the other.

 Dear Gianmichele Massimo ,

As, I see from the gel image, you could increase the template concentration and could go lower annealing temperature of different gradient. 

Good luck!

I am trying to amplify my desired gene from Genomic DNA. Initially i standardised it and replicated it. again I isolated Genomic DNA from blood and tried to amplify it to send it for sequencing but i am not getting any band at the standardised annealing temperature. How is it so?please help me out

Hello Megha

There are much Troubleshooting see on attchment 1

https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=12&cad=rja&uact=8&ved=0ahUKEwic2JXQlc_WAhWjQJoKHY7sDiAQFghYMAs&url=https%3A%2F%2Fwww.fws.gov%2Faah%2FPDF%2FPCRTS1_NoBand.pdf&usg=AOvVaw2tCl7T6kbx5AcfljMu7GAd

2

https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=13&cad=rja&uact=8&ved=0ahUKEwic2JXQlc_WAhWjQJoKHY7sDiAQFghdMAw&url=https%3A%2F%2Fwww.neb.com%2Ftools-and-resources%2Ftroubleshooting-guides%2Fpcr-troubleshooting-guide&usg=AOvVaw3KZmOyiiq1OH4MoZEjOLek

3

https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=14&cad=rja&uact=8&ved=0ahUKEwic2JXQlc_WAhWjQJoKHY7sDiAQFghjMA0&url=http%3A%2F%2Fbitesizebio.com%2F343%2Fthe-essential-pcr-troubleshooting-checklist%2F&usg=AOvVaw0PvOzYJf0s2jtWzBCbZntZ

4

https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=15&cad=rja&uact=8&ved=0ahUKEwic2JXQlc_WAhWjQJoKHY7sDiAQFghqMA4&url=http%3A%2F%2Fwww.bio-rad.com%2Fen-us%2Fapplications-technologies%2Fpcr-troubleshooting&usg=AOvVaw0-ZLDZEJ9UBbPqxvz-SZ0Q

5

https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=17&cad=rja&uact=8&ved=0ahUKEwic2JXQlc_WAhWjQJoKHY7sDiAQFgh3MBA&url=http%3A%2F%2Fwww.sciencedirect.com%2Fscience%2Farticle%2Fpii%2FS2214753514000102&usg=AOvVaw3d4rLNP1HPdiAmm5XXpsE_

6

https://www.genscript.com/pcr-troubleshooting-guide.html

7

https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=19&cad=rja&uact=8&ved=0ahUKEwic2JXQlc_WAhWjQJoKHY7sDiAQFgiEATAS&url=https%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fpmc%2Farticles%2FPMC3573865%2F&usg=AOvVaw26LOQQHjVt_2S-G62qfglY

8

https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=11&cad=rja&uact=8&ved=0ahUKEwjE-PX5l8_WAhWIPZoKHcYDAv44ChAWCCYwAA&url=https%3A%2F%2Fwww.scientistsolutions.com%2Fforum%2Fdna-pcr-real-time-pcr-standard-polymerase-chain-reaction-pcr%2Famplification-gut-contents-not&usg=AOvVaw3kHtfXScDahu2I-CzKBBE0

9

https://www.qiagen.com/be/resources/molecular-biology-methods/pcr/

10

https://palumbi.stanford.edu/SimpleFoolsMaster.pdf

11

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12

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13

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14

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15

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16

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17

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18

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19

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20

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My colleague ask this question

I am working on cDNA-SCoT , but i got smear with the band

Hi Nidaa, please, reduce your template concentration. For cDNA as template you could reduce to 100ng, 150 ng & 200ng Concentration.

Good luck!