how to add flag to a vector - (Oct/25/2003 )
I want to add a flag tag to the pAc5.1 drosophila expression vector, I use Kpn1 and EcoR1 to sequentially digenst the vector in 37c for 1 hr, and then ligation it use invitrogen T4 ligase 1U in 20 ul reaction and incubate at room temperature for 1hour, then transfome the DH5-alpha,
but the control contain only digested vector and T4 ligase ( no flag insertion) grown the same number colonies as those with insertion.
can any one help me?
(flag tag is obtained by synthesis two oligonucleotide about 35bp and then mix them and annealed)
Kpn1 has a 3' extention while EcoR1 has a 5' extension i suppose that you designed the oligo's to complement these sites correctly.
Then what might work for you is to take a bit of your gel purified vector (no Cip) and then add your annealled unphosphorylated oligos.
Set up several ligations. Keep the vector amount constant add different amounts of your annealed oligo's. Ligate o/n.
Start with a very high concentration of oligo's (from almost as high as you can go).
Under these conditions the vector is likely to ligate two oligo's one at each end. It is then not able to self ligate anymore. So if you use much oligo you should get only a few clones while with very little you should get more.
OK, this is only the case if the vector can self ligate. In anycase. like this you can only ligate one Flag-tag. The less clones the better since they are more likely to have the tag (unless of course you have undigested vector in your sample). 1 in 6 or 1 in 12 should have the insert like this, and that is if the vector can self ligate.
I suppose you can try a simmilar strategy in your case since you should be able to get rid of much back ground if say, you have some fragments with both ends Ecor1 or both ends Kpn1.
Don't know might be worth a try.
Just to check- you did phosphorylise your annealed oligos before trying to ligate them to the vector?? If not, then you won`t get any +ve colonies!!
Its not true that you will not get any clones if you do not phosphorylate your primers (provided that you do not phosphorylate your vector, and the anealled oligos overlap over a fair number of nt).
If you do phosphorylate them you have to find the right concentration to get one insert of the flag tag, and fish it out of the back ground.
That could work fine too. But if you do it the other way you should get fewer clones when u use much linker (so you know something is working, and you can't get two inserted flag tags). So if you pick some clones from a plate with high concentration of primers you should find one with the flag-tag.
Or maybe it is all luck.....
and now ,I have get many clones with low background ,and the digestion and pcr result is shown the right pattern, and the plasmid is now under sequencing.
thank for all your kindly help.
So out of curiousity what did you do (differently) in the end to make it work?
PS in my last post first sentence in between brackets it should read "dephosphorylate" not "phosphorylate", sorry)
I am soory to delay this replay for so many days.
In those depressed days, I find a similar protocol in Ambion which is to construct the siRNA expression vector, for it is also to insert about 50bp oligonucleotides into the vector.
I use the annealing buffer as follow
10X Annealing Buffer: 100 mM Tris-HCl, pH 7.5, 1 M NaCl, 10 mM EDTA.(I got it form google,not the same as describe in the Ambion mannual,)
1.add the two oligonucleotides 100ng each to a 50ul total volum ,
2.and heat at 95 for 5min, and slowly cool to 75c and keep at this temperature for 30min.
3.put the tube in a cup contain 70c hot water and put the cup on the bench to let it slowly cool down to room temperature
add about 50ng of well double digested and gel purified vector.
add about 8ng of annealed oligonucleotide
add the ligation buffer
add ligase 1U (invitrogen)
stay at room temperature for 30min
then use 2ul of ligation mixture to transform the E.coli.
(And it is not necessary to phosphorylate the oligo. the host will repare the gap.)
( the vector must be gel purified,though in my vector the fragment is only 20bp,but when I use NaAc and Ehtonal ppt, I got a very high background)
(my English is not very good ,wish you can understand it)