plant genomic DNA library preparation - (Jul/19/2007 )
Hello Guys,
I would like to prepare a plant genomic DNA library and have a big mess in my head… There are some questions…
1. The best methods for plant gDNA isolation for library preparation?
2. What vectors do you use for plant genomic library preparation?
3. What method for DNA fragmentation do you use?
4. Is size fractionation needed?
5. Is it easy to recover digested DNA of up to 10 kb from an agarose gel?
6. How much DNA do I need for digestion and effective recovery from a gel?
7. Have you been ever preparing a plant genomic library in pUC or similar cloning vector, inserts of what size were efficiently cloned?
8. Have you been trying to prepare a plant genomic DNA library in a TA vector (DNA digestion, AAA addition, cloning into TA and bacteria transformation)?
And one more point…
Southern analysis with a probe which represents a 3’ cDNA sequence of my gene gives three bands in every of three digestions (HindIII, EcoRI, BglII) – we suppose there are three paralogs in a genome. Should I prepare a library from size fractions of HindIII, EcoRI or BglII digestions that correspond to bands from Southern blot and then pray that the positive clones have more than a 3’ end or better prepare a library from partially digested DNA...
Of course I am thinking about a cheap and efficient strategyJ
Thank you for any suggestions and sorry it took so long…
MOI
I would like to prepare a plant genomic DNA library and have a big mess in my head… There are some questions…
1. The best methods for plant gDNA isolation for library preparation?
2. What vectors do you use for plant genomic library preparation?
3. What method for DNA fragmentation do you use?
4. Is size fractionation needed?
5. Is it easy to recover digested DNA of up to 10 kb from an agarose gel?
6. How much DNA do I need for digestion and effective recovery from a gel?
7. Have you been ever preparing a plant genomic library in pUC or similar cloning vector, inserts of what size were efficiently cloned?
8. Have you been trying to prepare a plant genomic DNA library in a TA vector (DNA digestion, AAA addition, cloning into TA and bacteria transformation)?
And one more point…
Southern analysis with a probe which represents a 3’ cDNA sequence of my gene gives three bands in every of three digestions (HindIII, EcoRI, BglII) – we suppose there are three paralogs in a genome. Should I prepare a library from size fractions of HindIII, EcoRI or BglII digestions that correspond to bands from Southern blot and then pray that the positive clones have more than a 3’ end or better prepare a library from partially digested DNA...
Of course I am thinking about a cheap and efficient strategyJ
Thank you for any suggestions and sorry it took so long…
MOI
Hi... MOI
during my final year project for my 1st degree, i had constructed a genomic library for oil palm. Following are the answers for your questions based on my experiance:
1.CTAB by Doyle and Doyle
2.phage
3.partial digestion
4. i didn't determine the size
5.yes, you may run your sample together with 1kb ladder
6. the standard volume would be 5 microliter but you must also run your gDNA to roughly determine the concentration or perform the OD reading. If the band looks faint, use 10 micoliter instead. Otherwise, if it is too thick, try 3 microliter. but 5 would be ok.
7 & 8. No, sorry