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Sequential restriction digestion of plasmid


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#31 friendly

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Posted 27 February 2009 - 04:18 AM

Posted Image

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

Edited by friendly, 27 February 2009 - 04:19 AM.


#32 T C

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Posted 27 February 2009 - 05:17 AM

Hey

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

Best
TC

#33 NemomeN

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Posted 27 February 2009 - 04:06 PM

The DNA looks fine...it'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.

#34 phage434

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Posted 27 February 2009 - 05:54 PM

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

#35 friendly

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Posted 27 February 2009 - 08:07 PM

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?
thanks

#36 phage434

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Posted 28 February 2009 - 05:18 AM

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.

#37 friendly

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Posted 28 February 2009 - 11:03 AM

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 ..

#38 phage434

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Posted 28 February 2009 - 01:03 PM

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.

#39 friendly

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Posted 01 March 2009 - 08:19 PM

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

#40 friendly

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Posted 02 March 2009 - 05:55 PM

Posted Image

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?

Thx

#41 phage434

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Posted 02 March 2009 - 08:26 PM

This looks good to me. The size range appears to be 1Kb - 30 Kb or so. For 50% gc dna, EcoRI will cut about every 4Kb on average. With high GC content (I assume) of banana DNA, the average fragment length will be substantially longer, in agreement with your picture. As I said earlier, you can make a library from this, but it almost certainly will not be complete. Depending on your goals, that could be ok. Some of the fragments are likely not to be clonable in high copy E. coli vectors, if that is your next step, and if that matters.

#42 friendly

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Posted 02 March 2009 - 10:04 PM

Hi Phage, Thanks for your prompt reply.
Do you think I need to use other enzyme to digest the DNA ??

#43 friendly

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Posted 04 March 2009 - 02:16 AM

Posted Image

This is my latest gel photo taken using a digital camera.
Lane 1 : 1kb ladder
Lane 2 = lambda DNA
Lane 3 = Lambda DNA with BamH1
Lane 4 = sample A's DNA
Lane 5 = sample A's DNA with BamH1
Lane 6 = sample B
Lane 7 = sample B with BamH1

Digestion were done in 37oC for 2 hours and I ran on 1% gel with 50V.
Apparently the lambda DNA with BamH1 is not the same as the photo in fermentas website. In fermentas website, their gel photo showed that BamH1 make four cuts on lambda DNA but in my sample I see five.
What make them different?

another question is how is digestion? suitable to proceed for the next step?
Phage, is it big enough for library this time?
thank you very much.

#44 friendly

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Posted 05 March 2009 - 07:26 AM

hihi...waiting for reply ..
please...

#45 phage434

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Posted 05 March 2009 - 08:04 AM

The inserts look perfectly prepared. Cloning them into a standard high copy number vector, such as pUC19 should be straightforward from here. You will get many clones, each containing thousands of bases of DNA sequence from the banana genome. You can sequence some of these. I still have not heard what the goal of doing so is. This is surely not the way, for example, to completely sequence the banana genome, but that might not be what you want to do.




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