Posted 18 January 2005 - 07:45 AM
I need Help!! Please!
I have to put a 3.5 kB fragment into a 6.5 kB vector.
The vector has a Km resistance gene.
I use invitrogen chemically competent cells.
This is what I do:
I PCR amplify the Insert- I have an Nhe1 site and an Asc1 site that is introduced by PCR.
The same is true for the vector.
I then sequentially digest the vector and insert with ASc1 and then Nhe1.
Then I run the sample out on a gel;gel purify using Q biogene-gel purification kit. I transform using 5 ul of ligation reaction
these are the results:
NO DNA added: 0 colonies
No insert added: 0
NO ligase added: 0
1ul vector, 8ul insert,ligase, ligase buffer: 0
So, basically I did not get any clones.
I was wondering if it is absolutely essential to purify after PCR and before digestion?
Any help is welcome.
Posted 18 January 2005 - 07:53 AM
In addition, try inactivating your ligase as this can inhibit transformation efficiency.
Also, cleanup your PCR product after digestion if you don't already do this.
Finally, try altering your vector:insert ratio.
Posted 18 January 2005 - 08:34 AM
Thanks for the prompt reply.
I know that the plasmid is linearized-but I have no certain way of telling that the insert and vector has been double cut since the restriction sites are too close to the ends.
Thats why I sequentially digest the vector and insert so that the chances that both enzymes have cut are higher.
Do you know how I can inactivate ligase?
I do cleanup the PCR product after digestion.
I wanted to know if I have to do so before the digestion ?
I will try altering the vector:insert ratio.
Any other suggestions?
Thanks in advance!
Posted 18 January 2005 - 02:24 PM
You can always do the (almost) sure-fire ligation into TA-TOPO vectors from Invitrogen and cut out your fragment from there if you continue to have problems.
Posted 18 January 2005 - 02:45 PM
Posted 19 January 2005 - 01:09 AM
Posted 19 January 2005 - 04:19 AM