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Troubleshooting restriction digest


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#1 starvingstudent

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Posted 07 July 2009 - 07:41 PM

Hello all,
I did a restriction digest to linearize two different pcr-2.1-Topo plasmids (2 different genes make up the inserts in the plasmids.). I used Xba I to linearize my plasmids (cuts only once) and purified my linearized plasmids using a Qiagen purification kit. I ran the 'cleaned' linearized plasmids on a gel and beside it ran their respective uncut plasmids. Here's a picture of my gel: First and last lanes are ladder, lanes 2 and 3 are a cut and uncut plasmid#1 and lanes 4 and 5 are cut and uncut plasmid#2. I need some help to figure out what's going on in this gel. To me, it looks like lanes 3 and 5 (uncut plasmid) are supercoiled b/c it should migrate farther and the one band in lanes 2 and 4 signify linear product (and linear products don't migrate as fast). Is this correct? I'm not sure about the slight faint band in lane 2 above the darker band, does this mean this plasmid isn't linear?

Any clarification would be most appreciated!

Thanks in advance
starving student

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#2 TRA

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Posted 07 July 2009 - 08:13 PM

Hello all,
I did a restriction digest to linearize two different pcr-2.1-Topo plasmids (2 different genes make up the inserts in the plasmids.). I used Xba I to linearize my plasmids (cuts only once) and purified my linearized plasmids using a Qiagen purification kit. I ran the 'cleaned' linearized plasmids on a gel and beside it ran their respective uncut plasmids. Here's a picture of my gel: First and last lanes are ladder, lanes 2 and 3 are a cut and uncut plasmid#1 and lanes 4 and 5 are cut and uncut plasmid#2. I need some help to figure out what's going on in this gel. To me, it looks like lanes 3 and 5 (uncut plasmid) are supercoiled b/c it should migrate farther and the one band in lanes 2 and 4 signify linear product (and linear products don't migrate as fast). Is this correct? I'm not sure about the slight faint band in lane 2 above the darker band, does this mean this plasmid isn't linear?

Any clarification would be most appreciated!

Thanks in advance
starving student

First I would say, try loading a little less of your digests on the gel. run around 100 ng. It should be easier to visualise. Looks like you do have some uncut plasmid in lane 2. Check you restriction enzyme is in date and you are using right conditions and quantities of plasmid etc. Maybe add a bit more enzyme and cut for longer (3 more hrs??). Uncut plasmid lanes look fine to me.




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