having difficulty in ligating 70bp dsDNA fragments - (Sep/04/2008 )

I got 6 clones to do and none of them is done after 6 days' work.

I just ordered the plus strand and minus strand oligos from invitrogen, all of them approxiamately 70bps long, flanked by SacI & Hind III

Then I annealed them into duplex by following the protocol:

1. resuspend the oligos with ddH2O in 200uM concentration

2.

reagent volume
TNE(Tris-HCL,NaCl,EDTA) annealing buffer 20ul
plus strand ssDNA 10ul
minus strand ssDNA 10ul

3. 95o C heatshock 2min
4. room temperature cool down 1h or more and store on ice, ready to use
5. so i believe the final concentratio is about 50uM or so. and i also diluted them and determined the concentration is roughly 3.00ug/ul

Then I tried ligating the duplex to a vector prepared by myself

preparation is like this:
1. miniprep the plasmid from clones i have done sucessfully
2. double RE treatment Sac I and Hind III in multi-core buffer(all purchased from promega)
3. 1% agarose gel electrophoresis and recylcle the vector with Gel Extraction Kit from QIAgen
4. dilute the Dna extract 200 times and mesure concentration with spectrometer
5. The recycled vector concentration is about 0.12-0.13ug/ul

ligation system:
1. the size of vector is about 8Kb and roughly calculation of its MW would be 660*8000, so if i add 1ul of my vector, the number of molecules in the solution would be 0.12/(660*8000)
2. the MW of the fragment is about 40000, if i add 1ul of the fragment , num of molecules would be 3/40000
3. so if i add fragment:vector in volume ratio 1:1 , the molar ratio would be (3/4000)/(0.12/(660*8000))=33000:1
4. because i do not want to dilute the fragment, so i did so in volume ratio 1:1

then i did transformation and I got very few colonies, on some plate i got 5 colonies(how lucky i am!) while on some plate i got one colony

But I believe all of them are false positive! because i did double digest and single digest and negetive control as well, found that digests were all done but i just can not see the 70bps band on 1% agarose gel. I know that it is because i did not get the 100% cut vector.

what do you guys suggest me to do right now? please let me know anything wrong with my protocol

i've decided to perform a CIP treatment tomorrow!
What do you say?
Thank you all!

Thank you swanny!

But will you please give me a little bit more details? Because I am not sure whether the ligation buffer is compatible with the RE buffer.I have to dilute a little bit or something else?

(1) Your ratio is way too high. While you might think this will make things better, in fact it inhibits the reaction you want. Since the concentration of insert is so high, each end of the vector will be ligated to a (probably different) insert. The vectors cannot be ligated, and once this happens, that vector fragment will not transform. Dilute your vector to around 10-20 ng/ul, and add equimolar insert to optimize the recircularization with the insert. Low vector concentrations favor reircularization rather than concatamers.

(2) If the vector has a third restriction site between the SacI and HindIII site, cut with that enzyme as well, which will reduce background.

(3) You may have more luck by avoiding the gel purification, if the cutting is good. If you do this, increase the insert ratio to 3:1 or 4:1 insert to vector molar ratio, to out compete the religation of the original fragment of the vector you cut out.

(4) Another approach is to use PCR to prepare your vector, which will largely eliminate the background from uncut vector. PCR with primers containing the two restriction enzyme sites in the primer (along with a 6 bp minimum 5' overhang). Cut with the two vectors, and ligate the insert.

Thank you for all you guys' suggestions! I will try.