Hi Nimanie,

In general there are two possibilities:

1. column based clean up of the DNA isolated with the CTAB method. You may loose some DNA on the column, so if the amount is limited this approach might be problematic.

2. addition of PVP (polyvinylpyrrolidone) which helps to remove polyphenols fom the plant samples.

Some additional general remarks:

- low ratios 260/230 often occur in samples with low concentrations of DNA (low A260 values)

- photometric measurement of DNA isolated by CTAB method is overestimating the amount of isolated DNA a lot, as CTAB also is detected at 260 nm. This means you need to evaluate the real amount of DNA by a different method, for example on an agarose gel compared to a dilution series of lambda DNA.

Hope this helps!

Good luck with your experiment!

Gertrud

further clean up is a good idea but first try amplifying less dna as this will mean less inhibition and possibly add a little more Mg as many contaminants remove Mg from the reaction mix

you can clean DNA with CTAB extraction,:

http://primerdigital.com/dna.html

but the best way, run gel-electrophoresis for DNA purification. Use low melted agarose and use this DNA with agarose in PCR.

Sybr Green use for DNA staining, and for visialization with blue light.

Dear Nimanie,

I had the same problem, especially with wild plant types! I highly recommend using column based clean up protocol, this should guarantee the DNA quality! Gertrud is quite right, DNA quantity will be in lower limits in such protocols, so pay attention please!

Best luck.

Have a look to the following procedure:

A combine procedure of CTAB and Genaid kit:

· A one hundred (100) mg of fresh leaves tissue were smashed with 700 μl of CTAB then the mixture was transferred to a 1.5 ml microfuge tube.

· RNase A was added in a volume of 5 μl to 200μl of GP1 (GP1 was mixed with RNase by gently pipetting).

· The RNase A and GP1 mixture were added to the microfuge tube that contains the extraction mixture then it was vortexed for 5 seconds.

· The extraction mixture was incubated at 60°C in a water bath for 10 minutes, and tubes were inverted every 5 minutes. At this step, the elution buffer (100 μl for each sample) was incubated at 60°C in the water bath.

· GP2 was added in a volume of 100 μl to each sample and vortexed for 5 sec. then it was placed on ice for 3 min.

· The solution was transferred to 1.5 ml filter column tube.

· The tube (with filter column) that contains the solution was spun by the microfuge at 8000 g for 1 min.

· The filter column was discarded and the supernatant was transferred to a new 1.5 ml tube.

· GP3 and isopropanol mixture was added in a volume of 1.5 times of the supernatant then the mixture was vortexed for 5 sec.

· A 700 μl of the supernatant was transferred into GD column tube then spun by microfuge (13000 rpm) for 2 min.

· The GD column was removed and the supernatant was discarded, then the GD column was put back again to the same tube. So, the rest of supernatant was transferred to the same GD column tube, and centrifugation step was repeated in the same conditions.

· The supernatant was discarded and the GD column (which contains DNA sample) was placed in a new 1.5 ml microfuge tube.

· The W1 solution was added in a volume of 400 μl and spun at 13000 rpm for 30 sec.

· A volume of 120 μl of washing buffer with 480 μl of ethanol was added then spun at 13000 rpm for 30 sec.

· The supernatant was discarded and the filter column was put back in the same tube then spun at 13000 rpm for 30 sec to dry the column.

· The filter column (which contains DNA) was transferred to a new 1.5 ml tube and 100 μl of elution buffer (which already has been incubated in water bath at 60°C) was added to each tube. It was left for 3-5 min to absorb entire the elution buffer then it was spun at 13000 rpm for 30 sec., finally, the filter column was discarded and the supernatant that contains the DNA was kept in the refrigerator.

Dear Nimanie

You must purify your DNA as per standard protocol undoubtedl, it’ll decrease the amount of DNA, but I’m sure you’ll get quality DNA. Moreove, go for DNA estimation by using lambda DNA instead of spectrophotometric observation, cause lamnda method is much precise and accurate.

Best at of luck !

Thank you all for your suggestions