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Advice on amount of starting template for subcloning


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#1 HawkeyeGrad

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Posted 11 March 2011 - 01:54 PM

Hi All,

I am performing some subcloning experiments in which I incorporated my target into a TOPO shuttle vector and am now using restriction enzymes in a double digest to cut out my desired target to get it into my destination vector. After the double digest, I ran the products on a 1% agarose gel (my target is 6kb) with Sybr Safe instead of ethidium bromide.

However, the gel looks like big bltohes of green stain at the top of the gel. What is interesting is that the 1k ladder also did not show up on the gel with any bands, only the big blotch-like green stain.

I suspect that it was the SBYr Safe stain so I'm asking if anyone has advice on the amount of starting material works well with SYBR Safe or with ethidium bromide. I was trying to avoid using ethidium bromidebut if I have to, I will. Any dvive on troule shooting SBYR Safe, starting material concentration, etc would be greatly appreciated.

Thanks
--HawkeyeGrad

#2 perneseblue

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Posted 12 March 2011 - 10:08 PM

it sounds like the DNA-gel did not run. The DNA ladder should have run.

You might want to check your
-gel box
-power pack
-gel
-buffer concentration

Make sure your equipment is working and that the buffers have been made to the right concentration.
May your PCR products be long, your protocols short and your boss on holiday

#3 phage434

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Posted 12 March 2011 - 10:16 PM

Did you make your gel with buffer (not water)? A gel made with water will often behave this way.

#4 HawkeyeGrad

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Posted 14 March 2011 - 08:53 AM

it sounds like the DNA-gel did not run. The DNA ladder should have run.

You might want to check your
-gel box
-power pack
-gel
-buffer concentration

Make sure your equipment is working and that the buffers have been made to the right concentration.


I am using 1X TAE buffer supplied from the manufacturer. What is interesting is that I can faintly see two bands corresponding to what I expect the sizes of the plasmid and my insert will be but no other bands especially for the ladder...




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