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problems with restriction digestion

problems with restriction digestion - Protocols and Methods Forum

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  #1  
Old 02-08-2008, 06:09 AM
chemophoto@gmail.com
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Default problems with restriction digestion



Hi all,
Would really appreciate some help!!
I am trying to clone a gene and this is giving me a lot of problems.
When I started with an alkaline lysis prep of the plasmid and did a
restriction digestion with BamHI and XbaI I was getting extra bands
that should not be there. I than did a Midi prep of the plasmid and
did two sequential digestions with BamHI and XbaI and my problem
disappeared. I then went ahead with my cloning and finally got some
transformants. I did an alkaline lysis prep of my transformants for
screening and did some restriction digestions and it seems I have a
few clones with the right profile.
So I went ahead and did a Midi prep (to get a pure prep) to
electroporate into my bug. After I did my midi prep I did a digestion
with BamHI and XbaI (sequential) and again I am seeing extra bands
that should not be there (the same bands I saw before).

I used a 20ul digest, 1ul restriction enzyme, 2ul buffer and 1ul of
plasmid DNA. I do not know the concentration of my plasmid DNA (didn't
nanodrop it yet). I used 1ul plasmid DNA based on the agarose gel
result of the midi prep. I digested it for 2hrs at 37 and did an EtOH
pption and did the second digest.

I am wondering why I am getting extra bands. Am I using too much
enzyme? But I have used this amount of enzyme in digestions of 20ul
before and it worked. Is there something else that I am doing
wrong??
I thought the reason why I got the extra bands before was as I was
using an alkaline lysis prep..which might have contaminants (RNA
etc).

Thanks a lot
Mary31
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  #2  
Old 02-08-2008, 10:00 PM
WS
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Default problems with restriction digestion

Hi Mary,

can you run your DNA preps on the gel, too?. A too long alkakline step
(some ptotocols say 5min) definitely will kill your plamsid
(partially) and cause it to wind up in some strange undigestable
conformation that may appear as extra band. For standards E.coli lab
strains, a few seconds definitely are enough and you should work on
ice and immediately proceed with neutralization.

Beware, too, of XbaI sensitivity to certain methylation patterns
possibly causing poor or partial digests.

Best regards,

Wo



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  #3  
Old 02-08-2008, 11:24 PM
Sudheendra Rao N R
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Default problems with restriction digestion

Hi,
Just to clarify!
How have to come to that conclusion that the bands u are seeing are extra?
sometimes insert might have restriction enzyme sites (i guess u are using
two enzymes on either side of MCS).
Try putting your insert virtually into the plasmid..using any plasmid
manipulation software (Vector NTI, DNAuser, plasmaDNA, Gentle etc)..cut it
with the enzymes you are about to digest it with (after circularizing
it)..and see how many bands u get (neb cutter is also okay)..
if you have done this job and still getting extra bands then
1. either your Enzymes are contaminated
2. of your mixture is receiving extra DNA from outside(check the digestion
components..especially water)
3. something else!!

sudheendra.

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  #4  
Old 02-09-2008, 03:16 PM
Michael Sullivan
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Default problems with restriction digestion

Another thing to keep in mind is how you incubate the digestion
reaction. Lots of people incubate digestioin reactions in a
waterbath. Be very careful doing this, especially with enzymes that
have star activity. In a waterbath, what often happens is that the
bottom of the tube is warm, but the top of the tube is cool. Water
evaporates from the reaction and condenses on the top, increasing the
concentration of enzyme, salt and glycerol in the digest. For small
volume digests this effect can be significant, and I've certainly
seen BamHI digestions exhibit star activity when incubated in a
waterbath. I prefer to carry out my digest incubations in a warm air
incubator: initial heat transfer to the reaction is slower, but there
is no problem with evaporation/condensation since warm air surrounds
the entire tube.

Mike


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  #5  
Old 08-27-2009, 07:31 AM
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Default Re: problems with restriction digestion

solution protocol
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Old 11-25-2009, 08:48 AM
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Question Re: problems with restriction digestion

Hi Mary,

I think there are some things you can use to reduce STAR activity. You could try it anyway. Not sure of the conc. low i'd say.

-1-(4-Bromo-2,5-dimethoxyphenyl)-2-ethanamine hydrochloride (1)

most of the companies sell it

kt
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Old 03-25-2011, 08:23 AM
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Default Re: problems with restriction digestion

Dear Mary,

I am digesting my plasmid pET25b and my PCR product with XbaI and BamHI. Firstly, i measured the concentration of my plasmid and my PCR product, so for example, if I use 1ug plasmid then the enzymes should be 1uL, so seems like 1ug for 1 uL. Thats why, measuring with nano drop is necessary.

I made 20 uL of reaction mixture. I used some uL of plasmid which is equal to 1ug plasmid, 1uL of BamHi, 1 uL of xbaI, 1uL of buffer (I used 0.5xK buffer Takara, it should be 2 uL but since 0.5 so I have to use half procedure), and add miliQ water until 20uL, incubate in waterbath 37C, over night.

What suheendra said is right, u should check with dna cutter (using dna cutters tool from google) before decided the restriction site that u will use. Because if inside of your gene contains the same restriction site with your restriction enzyme, it will cut your gene as well, so as the result, u will get extra bands.

Anyway, tomorrow I will check the result and run into ligation process. I'll let you know what the result is. I hope we can share experience, because I also will do a lot of some digestions experiment in advance.

Rgrds,
etin
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Old 01-04-2012, 01:00 PM
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Default Re: problems with restriction digestion

any solution ?
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