*** DNA and Southern Blot ***

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Dustfinger
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*** DNA and Southern Blot ***

Post by Dustfinger » Thu Aug 10, 2006 7:39 pm

Hi people,
Hope you can help me with my problem :

A piece of DNA (8000 bases) is cut with the restriction enzyme BglI and then with HpaII and finally with the mixture of both. The fragments are separated from each other , then a Southern Blot is done (again with these enzymes).

I want to write a sort of "map of restriction sites" for the 8 kb DNA fragment,
1 kb commensurating with 1 cm. How shall i do that ?

need the answer as quickly as possible, thanks !
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Post by sdekivit » Thu Aug 10, 2006 9:40 pm

you know thetotal length of the sample (8kb), so with a marker-lane of fragments with known lengths (100kb,150kb and so on) you can analyse the size of the fragments of the separate blots and combine this to get the restriction map.

First you only use restriction enzyme 1 and do a gel electrophoresis and Southern blot and analyse the length of the fragments (complete digest)

Then repeat with the second restriction enzyme.

Then use the mixture and think logically to connect the restriction sites in the correct order.

Here's an example of my cell biology course 2 years ago:

Complete digest all. We have a sample of circular DNA 10kb long.

Fragments with EcoRI: 6.9 and 3.1 kb
Fragments with BamHI: 10 kb
Fragemtns of mixture: 5.1, 3.1 and 1.8 kb

Construct the restriction map wioth these data. (shouldn't be too hard)

(I hope you can see know why it's important to know the length of the fragments of the seperate enzymes AND the mixture with this example)

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Post by Dustfinger » Fri Aug 11, 2006 8:33 am

Thanks for the answer but i don´t even know how a restriction map looks like....
I´ve attached the results for the restriction (Image1) and for the Southern Blot (Image 2) to this post. Perhaps you (or somebody else) could solve an example ? Then i should be able to do the rest myself, thx.
Attachments
image1.gif
restriction with BglI and HpaII results
image1.gif (2.88 KiB) Viewed 4923 times
image2.gif
Southern Blot result
image2.gif (2.28 KiB) Viewed 4921 times
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Post by sdekivit » Fri Aug 11, 2006 9:56 am

some kind of restriction map would be:

........BglI.............HpaII...BglI
...........|...................|........|
---------------------------------------------
---------------------------------------------

Just dra a DNA-strand and give the locations with right proportionalty in distance where the restriction sites are

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Post by Dustfinger » Fri Aug 11, 2006 11:23 am

Hey, thank you ! Now everything makes sense to me
(1 kb being a cm etc.). i didn´t know it was so easy.

Thanks !
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One last question

Post by Dustfinger » Fri Aug 11, 2006 11:50 am

One last question:
I´ve got the restriction map now, but how can i find the starting point and the ending point of transscription ?
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Post by canalon » Fri Aug 11, 2006 12:03 pm

You cannot without more information (like a southern blot using an oriA probe (supposing a fairly classical bacterial plasmid).
Last edited by canalon on Fri Aug 11, 2006 12:43 pm, edited 1 time in total.
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Post by Dustfinger » Fri Aug 11, 2006 12:10 pm

@Canalon
Have you seen the two images i posted earlier this day ? I´ve attached a third (result of Southern blot, the primer being the same as the one i multiplicated the piece of DNA with) but i wonder how you can find these two points then, coz the three images are all information i have.
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image3.gif
result of Southern blot, the primer being the same as the one i multiplicated the piece of DNA with
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Post by canalon » Fri Aug 11, 2006 4:53 pm

Maybe you should give us everything including the starting data you have.

You mean the probe is one of your primer? If then, just use your brain, it should not be too hard to figure out wher everything start. You are supposed to add on your map which fragment was the starting point.
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Post by Dustfinger » Fri Aug 11, 2006 5:06 pm

You´ve already got all the information I have, just look at all my posts in this thread, especially the first.

sdekivit wrote:some kind of restriction map would be:

........BglI.............HpaII...BglI
...........|...................|........|
---------------------------------------------
---------------------------------------------



My restriction map looks similar like this example of sdekivit. You mean I just have to tell the length of the fragment I marked red ? For starting point I mean ?
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Image

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Post by canalon » Fri Aug 11, 2006 5:12 pm

Your prob tells you which is supposed to the irst fragment of your map,. So basically it should be the first piece on your map.
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Post by Dustfinger » Fri Aug 11, 2006 5:57 pm

Ah that´s good. Now I can do everything !
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