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Plasmid miniprep w/ Glass fiber filter plates ????

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#1 tarquin



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Posted 17 August 2010 - 08:19 AM

We are currently trying out a method for high throughput plasmid miniprep using 96-well Glass fiber plates.

To do this we are following the protocols set out by both Millipore (http://www.millipore...ons/tech1/tn004) and Corning (http://catalog2.corn...alsp_an_017.pdf).

Both of these protocols use a method that involves 6M KI solution as a "bind solution". However this is causing real problems as it is impossible to read on a spectro/nanodrop. (huge peak at 250nm) and eluted DNA just floats out of the wells on an agarose gel (no matter how many washed with 80% Ethanol).

When we carried out a test with our plates using protocol or Qiagen solutions (without KI) we avoided the problem but the yield was very very low (<10ng/ul).
However, when we used Qiagen PB buffer (binding) as the "optional wash step" the yield went up to ~30-40ng/ul (still not excellent but an improvement). I believe Buffer PB does not contain potassium iodide (KI).

My questions are:
Is there any way to avoid the KI bind solution? What can we use instead?
Are glass fiber plates the best plates for this task?
Or is there another protocol that anyone knows of for Glass fiber plates that uses a different bind solution?

Many thanks

#2 NemomeN007



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Posted 17 August 2010 - 10:42 AM

NUNC sells something comparable...at the end of the day, most glass fiber plates are the same, but you will need to add some "binding" salt....usually, an equal volume of 6M GuHcl will solve your problems...add after neutralization from the classic alkaline lysis step. (ie. 100 GTE, 200 NaOH/SDS and 150 3MKoAC/5M acetate...once mixed and neutralized, add 450 ul of 6M GuHCl, mix and then apply the clarified lysate to your glass fiber plates...wash with Qiagen PB (which is most likely 4M GuHCl and 20% Isopropanaol, which is probably why you were getting higher yields due to the high concentration of GuHCl) and then wash well with 80% EtOH and elute with your buffer of choice.

Good luck.

#3 phage434



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Posted 17 August 2010 - 06:08 PM

The Qiagen patent says PB is 5M Gu-HCl and 30% ethanol. I completely agree with your suggestions. Likely the added PB bound whatever lysate DNA was left on the plate, leading to low but improved yield. Sodium iodide is a pain in many ways.

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