HI everyone!
I had a new problem in my PCR. I have run the PCR for 5 samples as a trial. Then when i run the gel, i can see very nice and clear bands. When i run back the PCR for my 45 samples, i cant see any bands on the gel. I did twice, but still no bands. Anyone have any idea on this, why i got no bands for PCR products? I used the samples for my trial and i followed all other procedures exactly the same!
Looking forward for reply as soon as possible!
Thank you!
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5 replies to this topic
#2
ocean_sky83
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Posted 24 March 2010 - 04:28 AM
Did u run a positive control?
You can use the same sample that yield a product in the first time, and using that as a positive control for the other 45 samples. If your positive control did not show up now, maybe it is because of your PCR settings, or during the preparation step, did u change any reagents? Did u store your DNA properly?
Maybe try again..
You can use the same sample that yield a product in the first time, and using that as a positive control for the other 45 samples. If your positive control did not show up now, maybe it is because of your PCR settings, or during the preparation step, did u change any reagents? Did u store your DNA properly?
Maybe try again..
#3
vaishu
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Posted 24 March 2010 - 07:02 AM
I used the same samples (5 samples) that yield a product in the first time. Now i run the PCR for another 40 together with that 5 samples earlier i used. The problem is now there are not even a single bands is visible for those 45 samples. The PCR settings is exactly the same with earlier settings. During the preparation step also, i did in sterile condition and accurate pippette techniques! I didnt change any reagents. The buffer, primer, dNTP, Taq, MgCl2 all the same with the earlier.
Yes, i do store my DNA properly. I stored it -20 degree celcius.
I'm really confused about the problem. I am a final year student and this problem occur on my final year project. Now for my project paper, i need to discuss why i didnt get the bands! I dont know how and what to explain this!
Please anyone could help me?
Looking forward for reply as soon as possible!
Thank you!
Yes, i do store my DNA properly. I stored it -20 degree celcius.
I'm really confused about the problem. I am a final year student and this problem occur on my final year project. Now for my project paper, i need to discuss why i didnt get the bands! I dont know how and what to explain this!
Please anyone could help me?
Looking forward for reply as soon as possible!
Thank you!
#4
swanny
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Posted 25 March 2010 - 04:48 PM
How many times have you tried to repeat the PCR?
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#5
Holsten
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Posted 26 March 2010 - 12:11 PM
You should always use a positive control in your PCR. There could be plenty of reasons why it didn't work, but you can not know for sure unless you have a positive control.
#6
ocean_sky83
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Posted 29 March 2010 - 04:13 AM
Maybe try to repeat PCR again?
Another reason I can think of is if your DNA concentration is low.. how about increasing your DNA template? when you take your dna out from -20 celcius, did u mix it before u pipette out your DNA?
Another reason I can think of is if your DNA concentration is low.. how about increasing your DNA template? when you take your dna out from -20 celcius, did u mix it before u pipette out your DNA?
