Test Results 1 (Merlin Preps)

vogeljm at esvx11.es.dupont.com vogeljm at esvx11.es.dupont.com
Mon Feb 28 18:20:36 EST 1994


In article <2kevvm$fvq at nuscc.nus.sg>, mcblab47 at leonis.nus.sg (Chia Jin Ngee) writes:
>
>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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