Protocol Online logo
Top : New Forum Archives (2009-): : Molecular Cloning

Sequential restriction digestion of plasmid - (Feb/21/2009 )

Pages: Previous 1 2 3 4 5 6 Next

This is my photo..
1st lane : 100bp ladder
2nd lane : Lamda DNA
3rd lane: Lamda DNA with ECoRI
4th lane : Banana DNA
5th lane : Banana DNA with ECoRI

Is this digestion normal ?

Thanks TC



I am not sure but it looks like that the DNA quality is very poor due to which you don't see sharp bands. Do you isolate a lot of protein with DNA? Besides it looks like yr DNA is degrading. Fresh DNA is always advisable.

Either the gel pic that you have taken is not good or there is some problem with yr gel as well as the bands aren't as sharp as I would like to see.

Do repeat the experiment with fresh DNA and check.

Don't know if this would help but this is what I interpret. :D


-T C-

The DNA looks's genomic DNA digested with Eco so there will be some smearing. The fuzziness can either be due to bad focus or too long between staining and imaging as DNA will begin to difuse through the gel.


Looks OK to me. You expect a smear when you digest genomic DNA. Next time it would help to run a 1000 bp ladder.


Thanks for all your reply..
meaning now I can progress to the next step??
Anyone did a genomic library before??
I also heard that it will sure get smearing when you digest Genomic DNA...any journal mentioned about this bfore?


You're ready to ligate these into a vector to make a library. What's your end goal? The required quality of the library depends a lot on what you intend to do with it. This will give you relatively short fragments (perhaps 8Kb, given the likely relatively high GC content). The coverage will almost certainly not be complete.


thanks phage...
My project is basically to construct a genomic library..How would I know I would be able to get the complete coverage??
thank you ..


Well, the banana genome size is 600 Mbp or so. If you have 6 Kb clones, then you will need 100,000 of them to cover the genome once. For good sequencing, you need 5-10x coverage, so think more like a million clones. I don't think you are prepared for that, but perhaps I'm wrong. Otherwise, if you are looking for a specific fragment, then there are likely easier ways to find it.


thanks phage434,
I will run another round by using 1kb ladder..hope it shows me the actual size...


This is my latest gel photo....
Lane 1 = MassRuler DNA mix
lane 2 and 3 were lamda DNA and Lamda DNA with ECoRI
Lane 4 = 5ul of Banana DNA
lane 5= 5ul DNA with ECoRI
Lane 6 = 7ul of DNA
Lane 7 = 7ul with ECoRI

Any comment about this gel photo.
Let me know please..
All DNA were total genomic library.

ps- phage434, can I continue with the next step?

Attached File

Pages: Previous 1 2 3 4 5 6 Next