4 replies to this topic

[charisma]

Hi
I attached the picture of my gel, PCR gDNA with 2 different primers. but I have no band! It's so weird.Just some smears at the bottom!   I used to have nice sharp bands before..Is it PCR problem or what? The last lane is the gDNA itself. It's not the PCR product. And whats your idea about the gDNA?(I extracted it from rice leaves)

Looking forward for your comments.

Cheers,
Charisma

Attached Thumbnails

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[pDNA]

it looks like if you have a lot of RNA contaminationg your gDNA preperation. Do you performed a digest using RNase A or something like that?

You should try to get rid of this RNA since it it makes problems during PCR ...and you are overestimating your gDNA concentration by spectrophotometric measurements? Therefore you use to low amounts of template DNA. Digest your gDNA and purify it by isopropanol precipitation. Add DMSO (up to 5%) to your PCR reaction to remove secondary structure and increase PCR performance (sometimes vendors do offer a special buffer for the amplification of genomic DNA). If you are not succesful you can try doing a 2-step nested approach ...this is sometimes nececessary. Also try different template concentrations!

Regards,
p

charisma, on 29 November 2010 - 05:29 AM, said:

Hi
I attached the picture of my gel, PCR gDNA with 2 different primers. but I have no band! It's so weird.Just some smears at the bottom!   I used to have nice sharp bands before..Is it PCR problem or what? The last lane is the gDNA itself. It's not the PCR product. And whats your idea about the gDNA?(I extracted it from rice leaves)

Looking forward for your comments.

Cheers,
Charisma

[charisma]

pDNA, on 29 November 2010 - 11:10 AM, said:

it looks like if you have a lot of RNA contaminationg your gDNA preperation. Do you performed a digest using RNase A or something like that?

You should try to get rid of this RNA since it it makes problems during PCR ...and you are overestimating your gDNA concentration by spectrophotometric measurements? Therefore you use to low amounts of template DNA. Digest your gDNA and purify it by isopropanol precipitation. Add DMSO (up to 5%) to your PCR reaction to remove secondary structure and increase PCR performance (sometimes vendors do offer a special buffer for the amplification of genomic DNA). If you are not succesful you can try doing a 2-step nested approach ...this is sometimes nececessary. Also try different template concentrations!

Regards,
p

charisma, on 29 November 2010 - 05:29 AM, said:

Hi
I attached the picture of my gel, PCR gDNA with 2 different primers. but I have no band! It's so weird.Just some smears at the bottom!   I used to have nice sharp bands before..Is it PCR problem or what? The last lane is the gDNA itself. It's not the PCR product. And whats your idea about the gDNA?(I extracted it from rice leaves)

Looking forward for your comments.

Cheers,
Charisma

Thanks for your comment.I have another question.RNA contamination in plasmid extraction,also, makes problems during PCR? the same as u mentioned about gDNA.

Whats ur idea of increasing Mgcl...is it useful to get the band?

[pDNA]

i never change the MgCl concentration ...i just do gradients with different annealing temperatures.
So if you have a gradient machine this is as well advisable to use it for PCR optimization.

But first of all try to estimate your real template concentration, then use the exact concentration your polymerase manual suggests for gDNA templates, use the right buffer for gDNA templates and eventually use DMSO (according to the manual of your polymerase) ...if your reaction fails i would try to optimize the annealing temperature ...but first of all i would ensure the quality and concentration of your template DNA.

Regards,
p

[charisma]

Thanks a Ton!