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

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.

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

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.

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

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.

Back to my orignal question can you see plasmid DNA seperation at 1% agarose? I am trying to run just the plasmid itself and only see one band. Shouldnt i see 3? I see virtually the same band for digested and undigested plasmid. Any thoughts/?

phage434 on Mar 6 2009, 12:04 AM said:

Thanks Phage for the reply.
The photo I show u in lane 3 and 4 is DNA from the Dragon fruit. Apparently I didn't know what is the genome size for Dragon fruits so will u suggest me to continue? Will it be a full sequence. ? I doubt that because nobody have discover the total genomic size of it.
My goal is to construct a genomic library first before doing further action on it. Might need to look for functional gene from the library.

please reply and thanks again.

any idea phage?
I have been wondering should I continue as the Kit for Library is costly ..

Why do you need a kit? The cost of a kit will be very low compared to the cost of sequencing any substantial number of clones. Where are you going with this? What is the end goal?

phage434 on Mar 6 2009, 12:04 AM said:

I have to agree with phage434. This is most certainly not the way to construct a library.With fragment sizes that small (several kb), your library will required a hundreds of thousands of colonies to have a minimum of 3x coverage. If you are gene hunting, eukaryotes have introns, lots of them which can be really big. Fragments sizes of kb may not be able to able to capture your gene.

A cDNA library would probably be of more us. What is the project's objective?

If you are building a genomic library, it would be better if you used BAC to carry inserts 50kb to 150kb size range. Then your library need only contain several tens of thousands of colonies to have minimum coverage. You will need to do a partial digest on your genomic DNA. Then run out the partially digested DNA using pulse field gel electroporation.

(Do note that Ecoli doesn't like certain sequences. And if you are using restriction digest to fragment your DNA, DNA that doesn't have the restriction site will be under represented as ligated molecules and thus absent from the library.)

As for the Dragon Fruit... well as Phage434 has been saying, what is your purpose? If you are gene hunting, you could continue. Just screen a large number of colonies. With sufficient brute force and ignorance, you should be able to find your gene.

As phage434 has mentioned, you don't need a kit. Just the right vector. And screening this big library and maintaining it (if required) would be a lot more costly than the kit.