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Random library using TOPO vector


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#1 relapse71

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Posted 20 August 2009 - 06:55 PM

These are two gel pics. They are miniprepped clones I made a while
back, thought I'd give them a look and send off the ones with inserts.
Problem is, a majority of the clones have inserts of the same size, or
two sizes overall. My hope was, I could add to my data, possibly
finding a repetitive clone. In Gel 1, there are two clones that I think
have an insert (described below). In Gel 2, only 4 lanes have a
different insert size, or are blank. My guesses are: TOPO is ligating
to itself or some other phenomenon.


Pic 1:

Lane 1 is a kb ladder
Lane 2 in TOPO
Lane 3 is TOPO control cut with ecorI 750 bp insert
Lane 4-9 are TOPO vectors that are suppose to have inserts, selected
from white colonies. The inserts were made by randomly shearing genomic
DNA. Then ligated into TOPO. Lanes 4 and 7 have an insert. What about
the other lanes? They have an insert, but all are the same size.
Impossible. What is happening here.

Same with Pic 2:
Look at all the same sized inserts (of two various sizes). I count maybe
two real inserts, and some blank lanes. What is going on here?

Attached Thumbnails

  • pcr07.21.09.jpg
  • 08.20.09.jpg

Edited by relapse71, 20 August 2009 - 07:17 PM.


#2 relapse71

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Posted 24 August 2009 - 11:05 AM

Maybe I am not being clear. I sheared genomic DNA, repaired it, and ligated into TOPO. Insert sizes should vary because they are random. Look at the pics of the clones digested with ecorI. Most have an insert size of the same size. What are these inserts? My guess is the vector ligates to itself.

Pic 1:

Lane 1 is a kb ladder
Lane 2 in TOPO
Lane 3 is TOPO control cut with ecorI 750 bp insert
Lane 4-9 are TOPO vectors that are suppose to have inserts, selected
from white colonies. The inserts were made by randomly shearing genomic
DNA. Then ligated into TOPO. Lanes 4 and 7 have an insert. What about
the other lanes? They have an insert, but all are the same size.
Impossible. What is happening here.

Same with Pic 2:
Look at all the same sized inserts (of two various sizes). I count maybe
two real inserts, and some blank lanes. What is going on here?

#3 Penguin

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Posted 26 August 2009 - 04:48 AM

Mmmm not sure whats going on but here are a couple of ideas:

Maybe only 1 or 2 regions of of your sheared DNA are being preferentially inserted into your vector????

How are you shearing your genomic DNA? Could this be generating products of a single size?? Maybe your insert:vector ratio/ligation procedure selects for a particular size of insert???

Just guesses, can anybody else come up with any other suggestions?

P

#4 Warren

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Posted 03 September 2009 - 10:55 PM

Those mystery lanes are not TOPO clones, the "vector" isn't even running the right size. It looks like you have some contamination....its been a while since I have used the marker set you are using, but it almost looks like it runs about where partially digested pUC19 would run. It definitely looks like plasmid contamination of some sort. The topo vectors I have used have two antibiotic markers.....if you still have your ligations, just transform them again and use kanamycin, if its plasmid contamination and the plasmids are ampR, you should get much better results (although fewer colonies <_< Warren..

Maybe I am not being clear. I sheared genomic DNA, repaired it, and ligated into TOPO. Insert sizes should vary because they are random. Look at the pics of the clones digested with ecorI. Most have an insert size of the same size. What are these inserts? My guess is the vector ligates to itself.

Pic 1:

Lane 1 is a kb ladder
Lane 2 in TOPO
Lane 3 is TOPO control cut with ecorI 750 bp insert
Lane 4-9 are TOPO vectors that are suppose to have inserts, selected
from white colonies. The inserts were made by randomly shearing genomic
DNA. Then ligated into TOPO. Lanes 4 and 7 have an insert. What about
the other lanes? They have an insert, but all are the same size.
Impossible. What is happening here.

Same with Pic 2:
Look at all the same sized inserts (of two various sizes). I count maybe
two real inserts, and some blank lanes. What is going on here?






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