Problem with transformation - (Aug/28/2008 )

Dear all

I am trying to clone a gene of 2.36 Kb to pXMJ19 vector having size of 6.6 Kb… Tried different conditions. still no transformants…I am getting hundreds of colonies on the plate. But only having the vector sequence. The restrictions enzymes which I used are Hind III and Xba I. The antibiotic which is being used in the plate is chloramphenicol ( 10 µg/ µl)

The protocol which I used is as follows…

PCR
PCR purification
Restriction enzyme digestion of vector and insert – double cut, 37 degree celcius for 3 hours ( I had checked the activity of enzymes also and found ok)
Purification of digested products
Concentration checking
Ligation – vector : insert ratio…1:3.
Total reaction volume – tried 20 microlitre and 10 microlitre
Temperature: 3 different conditions; 4 degree, 16 degree and RT for overnight
Transformation: DH5alpha cells, using electroporation
After transformation, 800 µl of LB is added and incubated for 90 minutes at 37 degree celcius. The incubation time also I had tried from 30 minutes to 90 minutes
Plating 200 µl

If anyone could give me some suggestions to modify my protocol….I would be really grateful to you

Thanks in advance

Bindu Subhadra

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as you are getting hundreds of colonies on your plate of vector only. the problem may be your vector is not digested. try overnight digestion. you can digest the insert also for overnight. and use some controls so that it will be easier to figure out the problem. have you added extra sufficient nucleotide before the restriction sites in your primer? i think you should be aware of these things.

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Go to this section and read about the trick that Badcell uses, it could possibly help you

http://www.protocol-online.org/forums/inde...?showtopic=4947

Agree with party you should digest your vector overnight and dephosphorylate, I havent checked but do XbaI and HINDIII leave compatible ends ?

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Thanks a lot..I was waiting to get some clue to start off....i am going to keep overnight digestion...of course now only i actually thought about keeping a control...am very new to this cloning field. Hoping that things would be good this time...i had added three bases just in front of the restriction enzyme sites in the primer...anyway could i ask you one more thing...if the two sites in the vector are too close to each other, how far it will affect the activity of the enzyme...my enzymes are 6 bases apart....


Thanks a lot
Cheers

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Wow...thanks a lot for the link...it is quite informative...
My enzymes XbaI and Hind III produces different sticky ends...
This time i am going to do overnight digestion and try the protocol which badcell mentioned...

Cheers

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