What would be the main reason to cause such a smearing

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bbs_r
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What would be the main reason to cause such a smearing

Post by bbs_r » Wed Mar 29, 2006 2:16 am

What would be the main reason to cause such a smearing? (Check out the following picture)

Thank you!
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exp_1_280.JPG
This is a 2% agarose gel electrophoresis, I found a terrible smearing above and below the band
====================
Ladder --- A --- B
====================
A = Practical
B = Control
exp_1_280.JPG (5.77 KiB) Viewed 2361 times
Last edited by bbs_r on Wed Mar 29, 2006 3:18 pm, edited 2 times in total.

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LilKim
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Post by LilKim » Wed Mar 29, 2006 3:20 am

Well, i don't know what your template is ... or anything about your reaction (is this PCR? a digest???)

.... (so this is just a guess)

the heavier "smear" at the top of the gel may be genomic/plasmid (template DNA)

and the "smaller" smeary band (at the bottom of the gel) could represent small degraded DNA fragments, primers, dNTPS or... just fluorescent junk...???

So i'm guessing that the heavy band in the 3rd lane and the light band in the second lane represent your "desired" product??? (i'm guessing because you didn't provide enough info for me to know exactly)

If you're having trouble with smearing i strongly suggest you use a gel purification kit to clean up PCR... (quiagen has a simple column based kit that takes me less than 30 mins to gel purify 10 samples) ... it's pretty easy and fool proof...

good luck
- kim

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canalon
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Post by canalon » Wed Mar 29, 2006 4:13 am

Agree with LilKim: at the bottom, probably primer dimers and other DNA fragments. On top, High molecular weight DNA. On the right lane, way too much DNA to get a nice band.

And knowing what kind of DNA this was would help to diagnose your problem.
Patrick

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any proof. (Ashley Montague)

bbs_r
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Post by bbs_r » Wed Mar 29, 2006 8:56 am

Here is the recipe of my PCR:

For 2 reactions (i.e. one reaction total volume = 50 uL only):
------------------------------------------------------------------------
Buffer(10x) 10.00 uL
Distilled Water 87.20 uL
forward primer(336.4ug / 40.1nmol) 0.50 uL
reverse primer(321.84ug / 39.3nmol) 0.50 uL
dNTP 0.80 uL
Mouse cDNA Lib 50.00 uL (ethanol precipitation was carried out before exp)
Taq polymerse(5 units/ul) 1.00 uL
------------------------------------------------------------------------
Also, each reaction contains:
MgCl2(25mM) 3.00 uL
Last edited by bbs_r on Wed Mar 29, 2006 3:33 pm, edited 4 times in total.

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canalon
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Post by canalon » Wed Mar 29, 2006 12:42 pm

First thing your recipe is completely useless unless you do not give concentrations but, the question was were you DNA come from (PCR, Cell/bacteria extraction etc...).

So the conclusion since it comes from a PCR: white stuff below the 100bp band is made of the primers. As Lilkim said, you can get rid of them by any PCR purification kit.
The big fragments could even be some of your template. since I guess your library has been cloned in plasmids. If so, why not start with less DNA? Did you estimate the concentration of your template?
Patrick

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Post by bbs_r » Wed Mar 29, 2006 3:15 pm

recipe updated.

The lab supervisor just told me that the template I used was called "mouse cDNA lib", he didn't mention how this was made....

The reason to use this large amount of DNA is because the band only shows in this amount of DNA. i.e. this is the least amount of DNA I used to make this PCR work.

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Post by LilKim » Wed Mar 29, 2006 3:56 pm

Hey there

Use a spectrophotometer to determine the cDNA concentration and THEN set up your PCR correctly! If your starting DNA is too concentrated you CAN'T get a pretty gel (and there are situations in which you'll NEVER have a pretty gel even if everything is .. perfect ).

The key for making PCR work optimally:
- make sure your primers and template match
- Follow manufacturers suggested concentrations for EVERYTHING!!! (check their website or the insert in the box)
- Make sure your annealing temperature is good (too low = result in non-specific amplification... to high = no amplification)

Quite honestly, I wouldn't fixate on the appearance of the gel... as long as I know that the amplification conditions are good I make life easy... and gel purify.

cDNA libraries? (I was going to give you my definition but it's getting late) Check this out http://en.wikipedia.org/wiki/CDNA_library

I wish you the best!
- KIM

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