Methods Digest, Vol 47, Issue 16

manish mishra via methods%40net.bio.net (by manishjnu8 from gmail.com)
Thu Apr 23 02:37:55 EST 2009


Dear Wo,

As you have written

  "You might
> remember: the DNA (and other water soluble stuff) is in the phenol
> phase, lipids are in the chloroform phase and proteins are inbetween."

I am slightly confused. Is it really that DNA is in the phenolic
phage? I think when we isolate genomic DNA by  Phenol: Chloroform:
Isoamyl alchohol , the pH is around 8.

I think that nucleic acid should be present in aqueous phage at pH
8.The lower organic layer contains proteins dissolved by phenol and
lipid dissolved by chloroform.
I dont know at pH 8 whether RNA is in aqueous phage or at interface?

At lower pH the RNA is in aquous phage and DNA is in phenolic phage.

Am I correct. If possible kindly clear my confusion.

with regards
Manish



On Wed, Apr 22, 2009 at 10:34 PM,  <methods-request from oat.bio.indiana.edu> wrote:
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> Today's Topics:
>
>   1. phenol in alcohol precip (Ed Siefker)
>   2. Blue/white with pET15b, Rosetta2(DE3)? (oliver.starks from gmail.com)
>   3. Genomic DNA isolation from mammalian cells (Dwayne Taliaferro)
>   4. Re: Blue/white with pET15b, Rosetta2(DE3)? (DK)
>   5. Re: phenol in alcohol precip (WS)
>   6. Re: Genomic DNA isolation from mammalian cells (DK)
>   7. Expression and -N end rule (shifali  chatrath)
>   8. Re: Expression and -N end rule (WS)
>   9. Genomic DNA isolation from mammalian cells (clarification)
>      (Dwayne Taliaferro)
>
>
> ----------------------------------------------------------------------
>
> Message: 1
> Date: Tue, 21 Apr 2009 08:38:04 -0500
> From: Ed Siefker <ebs15242 from creighton.edu>
> Subject: phenol in alcohol precip
> To: methods from magpie.bio.indiana.edu
> Message-ID: <49EDCC3C.2000906 from creighton.edu>
> Content-Type: text/plain; charset=ISO-8859-1; format=flowed
>
> Hi.  I was a little hasty in doing some phenol/chloroform
> preps of genomic DNA and apparently didn't remove all
> the phenol before adding ethanol to precipitate.  How can
> I recover from this?
>
> I considered extracting with chloroform again, but I don't
> know if the phases will still separate with ethanol in there.
> Also, the DNA isn't in solution, so I'm guessing it would
> collect at the interface.  If there were more DNA there,
> I'd just fish it out with a pipette tip, but it's a small prep.
>
> Any thoughts?  Thanks a bunch
> -Ed
>
>
>
> ------------------------------
>
> Message: 2
> Date: Tue, 21 Apr 2009 07:26:50 -0700 (PDT)
> From: oliver.starks from gmail.com
> Subject: Blue/white with pET15b, Rosetta2(DE3)?
> To: methods from net.bio.net
> Message-ID:
>        <c86f0875-c90c-47ea-bf71-5d0b8b824659 from s31g2000vbp.googlegroups.com>
> Content-Type: text/plain; charset=ISO-8859-1
>
> I've got my gene inserted in pET15b, and I transformed Rosetta2(DE3).
> Colonies appeared in appropriate numbers on an Amp/Cam plate,
> indicating the cells must have the vector.  Without thinking it out I
> re-streaked a couple colonies onto another plate with IPTG/Xgal to re-
> test insert presence (I'm fairly confident in my original ligation but
> it's always possible that the cells from which I prepped my plasmid
> were cotransformed with empty pET15b as well).  These produced blue
> coloration.  But after thinking about it more, it seems like either
> blue should never be produced (regardless of insert presence/absence)
> or should always be produced (regardless of insert presence/absence) -
> because the insert is not disrupting a lacZ gene like it is in
> pBluescript or something of that nature.
>
> So...how's the blue being produced, and is it even evidence of my
> insert not being present?
>
> I'll be testing these with new preps and PCR or digest today or
> tomorrow anyway but thanks in advance for your help.
>
>
> ------------------------------
>
> Message: 3
> Date: Tue, 21 Apr 2009 13:20:43 -0400
> From: Dwayne Taliaferro <taliaferroD from mail.nih.gov>
> Subject: Genomic DNA isolation from mammalian cells
> To: <methods from magpie.bio.indiana.edu>
> Message-ID: <C61378AB.306E%taliaferroD from mail.nih.gov>
> Content-Type: text/plain;       charset="US-ASCII"
>
> Does anyone have a quick and reliable protocol for isolating gDNA from
> mammalian cells?  I  would be interested.
>
>
>
> ------------------------------
>
> Message: 4
> Date: Tue, 21 Apr 2009 22:59:52 GMT
> From: dk from no.email.thankstospam.net (DK)
> Subject: Re: Blue/white with pET15b, Rosetta2(DE3)?
> To: methods from net.bio.net
> Message-ID: <BdsHl.83185$9t6.49292 from newsfe10.iad>
>
> In article <c86f0875-c90c-47ea-bf71-5d0b8b824659 from s31g2000vbp.googlegroups.com>, oliver.starks from gmail.com wrote:
>>I've got my gene inserted in pET15b, and I transformed Rosetta2(DE3).
>>Colonies appeared in appropriate numbers on an Amp/Cam plate,
>>indicating the cells must have the vector.  Without thinking it out I
>>re-streaked a couple colonies onto another plate with IPTG/Xgal to re-
>>test insert presence (I'm fairly confident in my original ligation but
>>it's always possible that the cells from which I prepped my plasmid
>>were cotransformed with empty pET15b as well).  These produced blue
>>coloration.  But after thinking about it more, it seems like either
>>blue should never be produced (regardless of insert presence/absence)
>>or should always be produced (regardless of insert presence/absence) -
>>because the insert is not disrupting a lacZ gene like it is in
>>pBluescript or something of that nature.
>>
>>So...how's the blue being produced, and is it even evidence of my
>>insert not being present?
>
> Rosetta(DE3) is simply BL21(DE3)  with pRARE2 plasmid.
> The strain has WT lacZ, so naturally it makes blue colonies.
> It has nothing to do with your plasmid or it having an insert.
>
> DK
>
>
> ------------------------------
>
> Message: 5
> Date: Tue, 21 Apr 2009 14:09:10 -0700 (PDT)
> From: WS <novalidaddress from nurfuerspam.de>
> Subject: Re: phenol in alcohol precip
> To: methods from net.bio.net
> Message-ID:
>        <a4cb9874-cd47-477e-b2f5-f693c46b21d7 from h28g2000yqd.googlegroups.com>
> Content-Type: text/plain; charset=ISO-8859-1
>
> Dear Ed,
>
> don't worry. Just rinse your pellet a few times with EtOH (and spin
> down shortly to make sure you don't loose the pellet).. If you plan
> downstream processing with enzymes, make sure all phenol has been
> removed. You might dare to check thesmell; your nose should turn out
> to be a very sensitive GC system with a neuronal detector attached to.
> Chloroform might not be that good choice, even that DNA doesn't
> dissolve in it, (regardless if there is EtOH and or phenol present),
> as it does not dissolve phenol as well as ethanol does. You might
> remember: the DNA (and other water soluble stuff) is in the phenol
> phase, lipids are in the chloroform phase and proteins are inbetween.
>
> Have fun!
>
> Wo
>
>
> ------------------------------
>
> Message: 6
> Date: Tue, 21 Apr 2009 23:22:51 GMT
> From: dk from no.email.thankstospam.net (DK)
> Subject: Re: Genomic DNA isolation from mammalian cells
> To: methods from net.bio.net
> Message-ID: <8zsHl.69302$ua7.56187 from newsfe17.iad>
>
> In article <mailman.979.1240346807.13724.methods from net.bio.net>, Dwayne Taliaferro <taliaferroD from mail.nih.gov> wrote:
>>Does anyone have a quick and reliable protocol for isolating gDNA from
>>mammalian cells?  I  would be interested.
>
> The purpose of gDNA? For "high" yeild and reasonable purity,
> here is what I used on many different cells (dicty, insect,
> mammalian lines):
>
> Pellet cells from 10 ml culture, resuspend in 1% Triton X100,
> spin, keep pellet, lyse nuclei with 400 ul 1% SDS, 20 mM EDTA,
> add 600 ul chloroform, vortex 15", spin hard, take upper phase, repeat
> extraction 2X. Add RNAse to 0.1 mg/ml, let sit for 5', mix 3 volumes
> of Qiagen buffer GQ or 5 volumes of Qiagen buffer PB with one
> volume of chloroform-extracted material, load onto regular Qiagen
> miniprep column, wash 2X with PB, wash 2X with PE, elute with
> 100 ul of 1-10 mM Tris, pH 8.0-8.5 (or TE) heated to ~ 70C.
>
> DK
>
>
> ------------------------------
>
> Message: 7
> Date: 22 Apr 2009 02:45:33 -0000
> From: "shifali  chatrath" <shifalich from rediffmail.com>
> Subject: Expression and -N end rule
> To: <methods from magpie.bio.indiana.edu>
> Message-ID: <20090422024533.56211.qmail from f4mail204.rediffmail.com>
> Content-Type: text/plain; charset="ISO-8859-1"
>
> Dear all!
> I had mentioned my problem earlier also in the forum but nobody replied. I am sure you great minds should have a solution to my problem. Please help me if you can!
>
> I want to express a snake venom protein,~ 10kDa, 10 cys residues and 5 disulphide bonds.Mature protein has Arg as first residue after signal sequnece is clipped off.
> First of all, i tried yeast expression system.
> I expressed my protein in pPICZ alpha, under alpha factor signal sequence followed by Kex2 and Ste13 signal sequences, such that the mature protein will have native -N terminus with Arg as first residue, as of my protein. BUt, I could not see any protein expression even under different medium and proper aeration and whatever possible troubleshooting one could do.
> Recently, i read -N end rule which states that Arg is destabilizing residues and directs the protein for degradation to 26S proteasome.
> I checked with the company, they say, It hold true for every expression system.
> But, my question is, if that is the case, how the protein is stable in snake's venom. I have found the protein in the crude venom of the snake.
> Also, the protein is expressed under alpha factor signal sequence which is supposed to be secreted into the medium. Therefore,once the protein is out into medium, it is out of cell now, Kex2 and Ste13 cleavages should be happening outside the cell, therefore, there should be no proteasomal degradation. Am I right?
>
> After clipping off signal sequence, protein will be exported out, still will there be any proteasomal targetting of proteins with destabilizing residue at -N terminus?
>
> I read in -N end rule that, protein's life depends upon penultimate residue to Met. We know, for a protein to be synthesised, we need to have  a start codon,i.e. Met. In -N end rule they say, Met will be removed be Metaminopeptidase(MAP), so if we express a protein we should not be counting Met residue in the expected MW? Because, not all mature protein sequences start from Met.
>
>
> Then,I tried expressing my protein using E. coli epression system, cDNA cloned in pET19b in Rosetta gami cells and RIL cells.I couldn't see any expression. Cells were growing normaly.(I hope, the protein is not inhibiting transcription and translation). Met and Gly had to be added at -N terminus to maintain the reading frame.
> Protein is 10Kda and was cloned without tag sequence. I mean expected protein should be 10kDa.I confirmed the sequences before cloning and even plasmid isolated from ex-pression hosts. DNA is there but no protein.
>
> Please answer any, if not all, of the above question. i am struggling with all this for alst 8 months. Your suggestions and help will be highly appreciable.
>
> Thanks in advance.
>
> Shifali
>
>
>
> Shifali Chatrath
> Graduate Student
> Protein science Lab
> Dept. of Biological sciences
> National University of Singapore
> Singapore
> +65-65161210
> +65-96393449
>
> ------------------------------
>
> Message: 8
> Date: Wed, 22 Apr 2009 01:13:52 -0700 (PDT)
> From: WS <novalidaddress from nurfuerspam.de>
> Subject: Re: Expression and -N end rule
> To: methods from net.bio.net
> Message-ID:
>        <5db5bd9e-8539-425c-8f06-5da81a395164 from m19g2000yqk.googlegroups.com>
> Content-Type: text/plain; charset=ISO-8859-1
>
> Hi Shifali, maybe your protein is toxic? you could change codon usage
> and/or try higher eucaryotic cells.
>
> To narrow down the effect of signal sequences etc.: Have you
> considered putting your protein (i.e its cDNA) and/or part of it in
> front of a reporter gene like GFP or insert just anything known in
> order to disrupt a possile action?
>
> Best regards,
>
> Wo
>
>
> ------------------------------
>
> Message: 9
> Date: Wed, 22 Apr 2009 11:03:41 -0400
> From: Dwayne Taliaferro <taliaferroD from mail.nih.gov>
> Subject: Genomic DNA isolation from mammalian cells (clarification)
> To: <methods from magpie.bio.indiana.edu>
> Message-ID: <C614AA0D.30A6%taliaferroD from mail.nih.gov>
> Content-Type: text/plain;       charset="US-ASCII"
>
> I am looking for a protocol that will allow me to purify genomic DNA from
> mammalian cells with the purpose of using it for Southern blotting and PCR.
>
> DT
>
>
>
> ------------------------------
>
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> End of Methods Digest, Vol 47, Issue 16
> ***************************************
>



-- 
Manish,
Lab no 122,
SBT , JNU.



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