Types of Resin in Magic(Merlin) Minipreps

HEAZLEWOOD,Joshua BOTJLH at LURE.LATROBE.EDU.AU
Wed Nov 30 18:44:01 EST 1994


Hi all,
	This is another re-post. My last dealt with the Magic Mini Prep formula
where I alluded to another post about the types of diatomaceous earth used. I
got a few e-mail concerning this and would have dealt directly with them but I
accidently deleted the messages (oops!). So sorry to burden those not
interested and the news group with this post.

Josh

PS Following is a great example of how methods can be put through different
tests by different people on the net and then come together in a summary. A
real work saver.

RE-POST 
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Date: 23 Feb 1994 07:18:14 GMT
Organization: National University of Singapore
Keywords: Merlin Preps, silica gel, diatomaceous earth, plasmid prep

Hi to all in bionetland!

This is the first batch of test results for the Merlin Preps. This deals 
with the type of resin used.

The following absorption matrices were tested out:

Celite 545 (Fluka: 22140)
Celite as aid in Analytical Filtration (BDH: 33134)
Kieselgel 60 (Merck: 7729)
Kieselgel 60H (Merck: 7736)
KIeselgel 40 (Merck: 10180)
Silica gel for TLC (Aldrich: 28,851-9)

Note: The Kieselgels are also known as Silica gels in English edition 
Merck catalogs

All the matrices are suspended in Gu-HCl/KAOc solution from the Merlin 
Preps Reagents (1.5 g per 100 ml). Of all the matrices, the silica gels 
appear as a clear gel when settled at the bottom. Silica gel suspensions 
are almost invisible. Only Celite suspensions are visible.

The minipreps are performed as in the protocol and the results are as 
follows:

Celite 545 (Fluka)
Yield: seems less than standard miniprep (miniprep with phenol/chloro)
RE digest: ok
Sequencing: ok
Degradation: no degraded

Celite for Analytical Filtration (BDH)
Yield: Great! yield same as Kieselgel 60  (see below)
RE digest: ok
Sequencing: ok
Degradation: not degraded

Kieselgel 60 (Merck)
Yield: Best of all of them. EtBr flourescence in agarose gel is blinding.
RE digest: ok
Sequencing: ok
Degradation: not degraded

Kieselgel 60H (Merck)
Yield: ok about the same as Celite 545
RE digest: ok
Sequencing: ok
Degradation: no degraded

Kieselgel 40 (Merck)
Yield: average, don't know why. Less than Celite 545
RE digest: ok
Sequencing: ok
Degradation: not degraded

Silica gel for TLC (Aldrich)
Yield: Better than Celite 545 but slightly lower than Kieselgel 60
RE digest: ok
Sequencing: ok
Degradation: not degraded

I know this is difficult too see with words. For those who know how to 
uudecode, I may post a uuencoded scan of the agarose gel. Again this is 
only possible with a overwhelming response :-) Posting a scanned 
sequencing gel autorad is out of the question.

The vector used is a pBluescript SK- without an insert. Restriction 
digests are done with EcoRI. Degradation tests are done by incubating the 
plasmid dissolved in 1X PCR buffer for 48 hrs at 37C. 

Problems associated with the matrices above:

Celite 545 (Fluka): None
Celite for Analytical Filtration (BDH): None
Kieselgel 60, 60H, 40 and Aldrich silica gel: Tremendous column back 
pressure. My thumb and fingers nearly turned blue :-(

Ashok Aiya (ashok at biochemistry.bioc.cwru.edu) suggested the use of Celite 
577 (Fluka 22142). It is smaller and stays in suspension longer. I do not 
know if it can be better in performance than Celite 545, I didn't try it. 
It is also the same price as Celite 545. However, being smaller in 
particle size, it should perform better.

Kieselgel 60 HF254 graciously tried by Seppo 
(kaijalai at vikki.helsinki.fi), seems to have the same effect as the above 
silica gels. Instead of 1.5g/100ml, he resuspended 0.5g/100ml. The yield 
is still as good if not better. He also describes another save $ idea:

Instead of using Promega columns, a 1 ml syringe is pressed at the bottom
with a 1.5 X 1.5 cm Miracloth. The silica resin/matrix/DNA is passed thru
the syringe and washed (he uses a different wash condition, I will find
out from him). He then cuts the syringe at the 250 ul mark, cuts a hole 
on the cap of an Eppendorf tube, puts the syringe into it. Add the usual 
hot water/TE and spin it.

Conclusions:
The surprise here would be the Celite from BDH. At first, I didn't have 
much faith in it. I mean the Celite has this slightly off-grey/yellow 
colour. But it works. Also the reports that the Celite from Fluka not 
performing up to standards seems true. And the silica gels (except for 
Kieselgel 40) as expected worked the best. Celite 577 (Fluka) may another 
alternative, but it is not tested.

Recommendations:
The BDH Celite would be good enough for Merlin Preps. The silica gels are
best but high back pressure and being pricey could be a nightmare. I would
also suggest that all of you check your chemical storage cupboards for
such materials. They are very stable for zillions of years (that's what
one chemist told me) if kept dry; and you may have some that may negate
purchasing a new bottle. 

Also, weighing of such materials ought to be performed under a
chemical/fume hood. These things will wreak havoc in your lungs if
inhaled. 

What's next:
Currently, looking at a Gu-thiocynate based binding solution. I live near
the equator where it is summer since written history. Precipitating Gu-HCl
will never be a problem to me or anyone near the equator :-) So this
Gu-thiocynate based solution will benefit those in temperate climates. I
will post the results as soon as they come. When things are more or less
settled, I will post a new updated Merlin protocol.

Usual disclaimers apply. To all those who contributed, keep them coming
and thank you all. This study can't be complete without you ladies and
gentlemen. For those whose names aren't mentioned or are misspelled,
please forgive me. There will always be the next report. 

Blessings and regards.
--
Jin Ngee, Chia			Chemical Carcinogenesis Laboratory
(Genie, the OligoMan)		Institute of Molecular and Cell Biology
mcblab47 at leonis.nus.sg		National University of Singapore
Tel: (065)772-3797		Kent Ridge Crescent
Fax: (065)779-1117		Singapore 0511
"I'm Mr Chia not Ms Chia and I know Genie is a girl's name"-Jin Ngee, Chia




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