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PCR failed No band.. desperate for opinions.. gel image available


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#1 ocean_sky83

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Posted 29 August 2010 - 04:11 AM

Please view my gel image.. sorry for the "bright reflection", there wasn't any band. Only the top part of the ladder was a bit smeared. The first band of my ladder indicates 8 kb. The lowest band around 300 bp. I have stained my gel for close to 1 hour. I am also using 1% gel.


Additional details,
- My ladder is up to the maximal of 8kb.. the lowest band of the ladder is about 300bp..
- My product expected size is 4 kb.
- My electrophoresis run settings is at 100 volts for 80mins. My length of gel measured 8cm from the start of the wells to the end of gel..

- No product despite a few attempts :(

- PCR settings : 94 degrees celcius initial denaturation followed by
35 cycles of :
94 degrees denaturation for 30 seconds, 59 degrees annealing for 30 seconds, 68 degrees elongation for 4mins.
- I am using a ramp speed of 9600. There is an asterisk symbol seen in the cycle run. Every cycle the elongation time slightly extend a few seconds longer.
- I am using a new set of primers which I have never used before and I did do a "Blast" and it does shows that the expected product to be about 4 Kb.
- Melting temperature of my primers is 62 degrees. So for annealing temperature of primers I use 59 degrees.

- Because there is no result, I have tried the following to no avail...
1) increasing elongation temperature to 72 degrees instead of 68 degrees
2) increasing template DNA to 5ul instead of 4ul
3) using 40 seconds instead of 30 seconds for the primers annealing step
4) using 5 mins for initial denaturation instead of 2 min
5) using slightly more primers


I am using 50ul reaction per sample with 4ul of template DNA. My DNA is freshly extracted and I have used to run other PCR it is working. I don't have any problem. For my PCR mixture I am using the qiagen premixed... I have tried using the multiplex mix and also the standard pcr mix but both did not yield a product.

Help~~
Any advices greatly appreciated! Thanks!!

:)

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#2 Felipillo

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Posted 29 August 2010 - 07:08 AM

You have a lot of RNA, you need to treat with RNAse before genomic DNA extraction, and it seems like your DNA are degraded, so, you can do a DNA integrity gel, to known how the DNA looks after genomic DNA extraction.

Edited by Felipillo, 29 August 2010 - 07:10 AM.

Chance favors the prepared mind
Louis Pasteur.

#3 Ameya P

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Posted 29 August 2010 - 09:45 PM

Well Ocean,

I would suggest you try out different annealing temps. say from 54 C to 62 C for your PCR in addition to cleaning up your RNAs. The calculated temperatures are not necessarily the best temperatures of annealing.

Let me know how things work out.

Ameya

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