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tarquin's Content

There have been 5 items by tarquin (Search limited from 19-October 18)


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#86833 Guanadine HCl for plasmid miniprep

Posted by tarquin on 14 September 2010 - 03:23 AM in Molecular Biology

y cant you buy mini prep kit which is cheaper $107 for 50 minipreps qiagen


We have to do high throughput 96 well plates. Although the Qiagen plate miniprep is the cheapest here in Brazil, it's still bloody expensive. It's cheaper for us to buy Millipore plates and make our own solutions.



#86747 Guanadine HCl for plasmid miniprep

Posted by tarquin on 13 September 2010 - 09:29 AM in Molecular Biology

Hello

We have to buy GuHCl to use for high throughput plasmid minipreps before sequencing. Many commercial kits use the same chemical as a bind solution for binding DNA to the filter plate.

The problem is cost. 100g of molecular grade(99%) GuHCl is R$200, but this is enough for only 4 plates. Whereas 1KG of "technical grade"(98%) GuHCl is the same price yet enough for ~48 plates.

Do you think there would be a problem to use technical grade? Its for a 6M solution to add to the cell lysates following Solutions I,II, and III in standard miniprep.

Many thanks



#83831 Plasmid miniprep w/ Glass fiber filter plates ????

Posted by tarquin on 17 August 2010 - 08:19 AM in Molecular Biology

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



#82438 Miniprep DNA-strange nanodrop peak at 240-250nm?

Posted by tarquin on 04 August 2010 - 10:46 AM in Molecular Biology

It turned out to be the bind buffer (Potassium Iodide).
Thanks for your replies anyway.



#82043 Miniprep DNA-strange nanodrop peak at 240-250nm?

Posted by tarquin on 31 July 2010 - 03:41 AM in Molecular Biology

I have just started a high throughput DNA miniprep procedure in our lab using Millipore plates (I adapted it for use in a plate centrifuge as opposed to a vacuum manifold).
However I don't use a lysate clearing plate, instead I pellet the debris and transfer the cleared lysate as best I can to the DNA binding plate.
After 2 washes with 80% Ethanol the plate is spin dried for 10 min. I then elute with water, or TE.

On both occasions (TE or water elute) my nanodrop readings were strange. There was a sharpish peak between 240-250nm. There was still signal at 260nm, although this was the downward slope of the ~245nm peak, not a separate 260nm peak. Obviously my 260/280 ratio was high (2.2-2.7).

I've search the net to no avail. I'm thinking ethanol contamination, cell component contamination, or even something from the filter plates (spin speed too high?).
Please help!

Many thanks for your time.




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