Problems with directional cloning using BsmBI and Xba I - (Jul/20/2007 )
I'm hoping someone out there can offer some advice after reading this. I will try to be as clear and thorough as possible:
I am trying to clone a 550bp PCR fragment into 3.5kb pPICZalphaA (yeast expression vector). The restriction sites that I was able to utilize were BsmBI and Xba I.
My PCR procedure:
1. Designed primers for my DNA fragment with 6 bases before BsmBI recognition site (5' end + strand) and 6 bases before Xba I recognition site (5' end - strand).
2. Carried out PCR and observed a good amount of DNA of correct size.
3. Ran DNA on 0.8% agarose gel in TAE and excised fragment
4. Purified gel slice with Qiagen gel extraction kit - eluted with EB buffer.
5. Ran 1ul on gel to verify yield after purification - yield was good.
-I transformed my vector into GM2163 cells (dam-) since Xba I is sensitive to dam methylation and did a DNA prep using Qiagen miniprep kit.- eluted with EB buffer
1. Digested 1ug vector and 1ug insert sequentially with BsmBI first following NEB guidelines for 3h at 55C
2. Ran digested DNA on 0.8% agarose gel in TAE and excised fragments after verifying digestion had occurred.
3. Purified fragments by Qiagen gel extraction kit - eluted with 35uL EB buffer.
4. Ran 1ul of purified DNA on gel to verify presence of DNA - yield was good.
5. Digested 30uL of purified DNA with Xba I following NEB guidelines for 3h at 37C
6. Purified fragments by Qiagen gel extraction kit - eluted with 35uL EB buffer.
7. Ran 1ul of purified DNA on gel to verify presence of DNA - yield was good.
Quantitated DNA concentration by spectrophotometer:
vector: ~300ng/uL (~0.26 pmol/uL ends)
insert: ~150ng/uL (0.826 pmol/uL ends)
1:3, 1:5, 1:10 vector:insert ratios (did one set of reactions based on pmol concentration of DNA ends and the other set based on actual ng amounts)
Reaction volume: 10uL
1uL 10X Ligase Buffer
8uL Insert + vector + dH2O
1uL T4 DNA ligase
Incbuated overnight at 16C
Transformed 1uL from ligation reaction as well as 1uL of a 1:10 dilution of the ligation products (to make sure salt or ligase amounts would be sufficiently low as to not inhibit my transformation) into 50uL DH5alpha comp cells and plated on Low Salt LB + 50ug/mL Zeocin (pPICzalphaA vector selection marker)
and incubated in the dark at 37C overnight (~15h)
Also ran 5uL of ligation reactions on 0.8% agarose gel to see if ligation occurred - here are my results:
-please keep in mind I am estimating sizes based on 1kb DNA ladder from NEB
-self-ligation control (pPICZalphaA vector digested with BsmBI/XbaI and purified): 3.5kb, 4kb, 6kb and >10kb bands
-1:3 (both ligations) showed ~1kb and 8kb bands
-1:5 (both ligations) showed ~1kb and 8kb bands
-1:10 (both ligations) showed ~1kb and 8kb bands but also showed an additional band around 6kb
no DNA - control: 0 colonies
1ng pPICZalphaA (uncut vector): >500 colonies
pPICZalphaA self ligation: 4 colonies
1:3 (pmol) ligation: 7 colonies
1:5 (pmol) ligation: 25 colonies
1:10 (pmol) ligation: 10 colonies
1:3 (pmol) diluted ligation: 0 colonies
1:5 (pmol) diluted ligation: 13 colonies
1:10 (pmol) diluted ligation: 3 colonies
1:3 (ng) ligation: 2 colonies
1:5 (ng) ligation: 15 colonies
1:10 (ng) ligation: 19 colonies
1:3, 1:5 and 1:10 (ng) diluted ligations: 0 colonies
And now - FINALLY - here is my question:
Do you think that I have ligation for my 1:10 reactions based on the agarose gel run after ligation?
Is it worth screening any colonies?
Any advice would be much appreciated - I have been trying to clone for 1 week now with no luck!
Thanks a million!!
why not sequencing it if you have done so much work, if you are uncertain you could cut some plasmids with the two enzymes and see if there are inserts there. I am not sure if I understand your quesion.
You have many fold more colonies in your ligation (1:10) than negative control. So you should go ahead and try to make DNA and do an analytical digest to confirm presence of insert.
go for it, it doesn't take long. As scolix has pointed out, it does seem that you have more colonies on the ligation plates. A colony PCR should be able to show if the 550bp fragment has been inserted.
I am waiting for my DNA to digest right now
My main question was whether I could eliminate some minipreps - I ask since it appeared that there may have been successful ligation only for the 1:10 vec:insert reaction based on the agarose gel.
Does anyone else check their ligations on a gel before transforming?
I did when I first started. I would consider it good practise.
However after a while, you kind of get the hang of ligation work, and you start dropping many of the controls and safety nets in exchange for speed.
I do not check ligations on gel before transforming. To me, its a waste of time.