Cloning Construction of 9000bp vector with 900bp - (Aug/28/2006 )
Hey, guys.
I have tried many times of making the new construction with 9kb vector with 9bp insert, but I never got positive colonies. This made me so so upset, would you mind giving me some constructive comments from you, please? Many thanks.
The work I have done:
Double digestion of plasmids with AgeI and BamHI
Ligation conditions: 1) 16 degree, overnight
2) 4 degree, overnight
3) Roomtemperature, o/n
Transformation condition: 1) Commertial competent cell, DH5 alpha
2) Normal transformation with 30min ice incubation, 45sec heat shock, also with 1hr LB incubation
I really don't know the reason, I think I was quite puzzled now...hope anybody of you could help me out
Thanks a million...
Could u verify that AgeI is digesting ur vector. Sometimes ageI doesnt work.
how long do u digest ur DNA. too long might also interfere.
Does ur vector (9000bps) have any possibility for recombination. If so, u need to use different cells for transformation.
how long do u digest ur DNA. too long might also interfere.
Does ur vector (9000bps) have any possibility for recombination. If so, u need to use different cells for transformation.
Well, I've checked the agarose Gel of my digestion, and I could clearly see the band which were cut buy both AgeI and BamHI
The digestion for my DNA: I set double digestion for 2.5hrs
I'm not sure about the recombination, does it interfere much? if possible could you give more information, please. Thanks
Recombination depends on the type of vector u r using. If ur vector has IRES or some repeat sequences for eg. in viral vectors, they r prone to recombine and using DH5-alpha doesnot help. U need to use recombinant nagtive strains like SURE cells or STBl3 cells to transform ur ligation.
Hmm…
Quite an irritating problem you have.
My assumptions.
- The 9bp insert is a synthetic (nonphosphorylated) oligo you have purchased.
- The 9kb vector has been checked, so that it is known that both restriction digest have gone to completion.
- The 9kb vector fragment has also been gel purified, taking care that the product is free from chaotrophic agent and the ethanol wash solution.
Have you tried this? (Forgive me if you are already doing this)
Use a very high concentration of your 9bp oligo, 10mM and maybe higher, with a small quantity of your 9000bp vector.
Maybe something like
15 ul 10mM 9bp oligo
3ul 9000bp vector
2ul T4 ligase buffer (freshness can be determine by smell)
0.4ul T4 ligase (T4 ligase does go off after some time even at -20 Celsius)
Ligate overnight at 16 Celsius.
Hope this helps.
What exactly do you mean with "no positive colony's"? Did you get colony's on your negative control (self ligation as well as non-ligated vector)?
Could't you try to do this with site directed mutagenesis principle? PfuUltra (or PfuUltra II) should be capable of amplifying 9 kb (I've gone up to more than 8 myself)? Easiest way of doing this would be to design primers , of which one has the extra 9 bp, buy them phosphorylated, do the PCR (12-20 cycles should do the trick), cut all with DpnI and then ligate all of it and transform competent cells. First need to check the PCR conditions of course.
Another way of trying would be to cut out your area of interst of the plasmid, clone into pUC or so, do the cloning and then get it back into your original vector.
I ligated 20 bp to a 9 kb vector ordering the a pair of compliemntary and phosphorylated oligos, after anealing of course they should have the free ends complementary to the free ends of your digested vector.
Using promega t4 ligase ON ligation at 4 I got enough colonies in the 1:8 vector:insert reaction. No colonies at all in the control (ligation of cut vector)
I insist that calculations are important
I have tried many times of making the new construction with 9kb vector with 9bp insert, but I never got positive colonies. This made me so so upset, would you mind giving me some constructive comments from you, please? Many thanks.
The work I have done:
Double digestion of plasmids with AgeI and BamHI
Ligation conditions: 1) 16 degree, overnight
2) 4 degree, overnight
3) Roomtemperature, o/n
Transformation condition: 1) Commertial competent cell, DH5 alpha
2) Normal transformation with 30min ice incubation, 45sec heat shock, also with 1hr LB incubation
I really don't know the reason, I think I was quite puzzled now...hope anybody of you could help me out Thanks a million...
I'm quite happy to see so many suggestions, Sorry, guys, that was my fault....
The size of the insert is 900bp. But I could not edit it after I posted this topic. I'm so sorry........
I've never used SURE and STBI3 cells before, I'll go and check more information on it.
And I tried Ultracompetent cell XL-10 gold cells, although I got some colonies, but after checking from gel, they are not the right ones...
Thanks.
Quite an irritating problem you have.
My assumptions.
- The 9bp insert is a synthetic (nonphosphorylated) oligo you have purchased.
- The 9kb vector has been checked, so that it is known that both restriction digest have gone to completion.
- The 9kb vector fragment has also been gel purified, taking care that the product is free from chaotrophic agent and the ethanol wash solution.
Have you tried this? (Forgive me if you are already doing this)
Use a very high concentration of your 9bp oligo, 10mM and maybe higher, with a small quantity of your 9000bp vector.
Maybe something like
15 ul 10mM 9bp oligo
3ul 9000bp vector
2ul T4 ligase buffer (freshness can be determine by smell)
0.4ul T4 ligase (T4 ligase does go off after some time even at -20 Celsius)
Ligate overnight at 16 Celsius.
Hope this helps.
Sorry, I input wrong size of my insert, it should be 900bp. I got two points, one is to get rid of ethanol which will inhibit the ligation. The other one is the molar ratio of the insert and vector,right? I think this also depends on the size of each fragment...
I'm sorry about this.