No colony for oligo cloning - (Jun/13/2009 )

Hello all, I am trying to get a new plasmid by inserting a 30bp dsDNA into a pBluescript based plasmid (~4.4kb). First, plasmid was cut by KpnI and XbaI sequentially (Ethanol ppt was used to change the buffer) which gave two fragments (~28bp and ~4.4kb). Then, agarose gel extraction was performed to get the expected band(~4.4kb) which was used in ligation with 30bp dsDNA. The 30bp dsDNA has been phosphorylated and used directly in ligation with Kinase involved (heat inactivated). The molar ratio was 1:20 (4.4kb:30bp). 1 ul NEB T4 ligase (2000U) was used in 20ul ligation solution. A pretty clear circular band was shown after ligation. 5ul ligation solution containing ~9ng circular band was used to transform 100ul competent cell, however, no colony was observed. I doubted the ampicillin may be too high, but no cology was shown after ampicillin concentration went down to 20ug/ml and 5ug/ml from 50ug/ml. I performed another transformation with a known plasmid purified by Qiagen maxi kit. Colony does show in this control experiment. Considering the impurity of ligation solution, ethanol ppt was done to the ligation solution to change the buffer into tris followed by transformation, but no colony again. So I doubt the digestion/ligation procedure has some problems. My boss said the UV exposure during gel extraction may cause transformation failure. That could be a reason. I am going to do the whole thing from digestion again. In order to avoid UV exposure, PCR purification will be performed after sequential digestion. Do you guys have any other suggestion? Many thanks.

Hi Zhongbo,

Can you visualize the 28bp band after digesting the plasmid? I think it is too small to be seen on agarose gel after plasmid cut.

How did you prepare the dsDNA? I guess it was made by annealing two ss-oligos, right?

Did you include no insert ligation control or no ligase control? Did they give the same circular band as seen from your ligation reaction?

How many colonies did you get from intact plasmid transformation?

Zhongbo on Jun 13 2009, 09:39 PM said:

i usually get such problems because of the following reasons:
1. the vector has not been properly digested by both the enzymes. so please make sure of that, giving 4-5 hrs of digestion time for each.
2. phosphorylation of the insert is not proper. furter, instead of Etoh pptating ur insert, you could try purifying it on Qiagen column.
3. the insert concentration is way too less.
4. uv does not seem to be that big a problem. i mean, it takes hardly 10 sec. to excise the band of ur interest. gel-extraction is how i normally get my vector with, n it works.
5. also make sure that ur ligase enzyme is working fine.

all the best.

pcrman on Jun 14 2009, 01:06 AM said:

Thank you pcrman,
28bp band can not be seen using NEB 2-log DNA ladder (0.1-10kb). Although this could be confirmed by using NEB low molecular weight DNA ladder (25-766bp), I didn't check that.
Yes, dsDNA was prepared by annealing two ss-oligos. Then phosphorylation was performed.
I did no insert ligation control but not no ligase control. The circular band does not show in the no insert ligation control.
For the intact plasmid transformation, 100ul solution (totally 1ml) was inoculated onto a 100mm plate with 50ug/ml Ampicillin. Each plate shows ~10 colonies. The plasmid has been purified and digested by enzyme, so it is confirmed that transformation is successful for the control transformation.

DRN on Jun 14 2009, 01:08 AM said:

Thank you DRN,
1, One ligation control with vector but not short dsDNA has been performed. There's no circular band shown. This may prove that the KpnI/XbaI digestion is successful since the vector can not be ligated by itself. This control experiment also proved that the ligase works well because there's a extra dimer band of vector shown. Monomer is ~4.4kb, dimer is ~8.8kb.
2, I don't quite understand "phosphorylation of the insert is not proper." You mean the short dsDNA could be ligated to the vector directly without phophorylation? Actually, I did ligation without phosphorylation of the short dsDNA first which showed the same bands pattern as the phosphorylated one. Since there's no colony after transformation, I did phosphorylation to avoid two nicks in the ligated product. But both gave me no colony.
3, What's the molar ratio you usually use for this purpose? You mean I should go to less than 1:20, such as 1:30 (vector:insert, 4.4kb:30bp)?
4, For the UV exposure, why do you think 10 sec matter?
Thank you so much.

I mean if you cut a 28-bp fragment off a plasmid, because the size is too small and thus the amount of DNA is too little to be seen on agarose gel.

When doing double digestion, you should do two parallel controls (cut the plasmid with individul enzymes) to make sure both enzyme work.

How did you anneal the two oligos? It could be very tricky. Make sure your oligo synthesis is of high quality (full length) and annealing is successful. There are several previous threads on this issue:
http://www.protocol-online.org/biology-for...posts/3579.html
http://www.protocol-online.org/biology-for...osts/27665.html
http://www.protocol-online.org/biology-for...posts/6399.html

When I do oligo cloning, I don't do extra phosphorylation of the oligos.

The number of colonies from positive control is a bit too low. You may need to use library grade instead of subcloning grade competent cells.

pcrman on Jun 14 2009, 12:05 PM said:

Thanks, the information is really valuable. I will run a control to confirm both enzymes work. The oligos are not PAGE purified, this worthes to try. I will report my results here later.

I suspect your competent cells are not very competent. You should get a lawn of transformants when transforming purified plasmid. To test competence, dilute plasmid to 10 pg/ul in TE. Transform with 1 ul of this diluted plasmid. You should get many colonies. If you get none, which is what I expect, you need better competent cells. 10**8 transformants/ug of DNA is a good and achievable target, which should result in 1000 colonies in this test.

I get colonies. Thank you all.
The reason why I failed previously is the UV exposure. This time, PCR purification instead of Gel extraction is used to get the longer vector since the short one is only ~30bp in length. For transformation, it works .
However, I get colonies on the control plate transformed by the vector selfligation. That problem may be due to not complete cut by the second enzyme (XbaI) or the shorter pieces of DNA residue after PCR purification. Anyway, one step closer.

I am trying to figure out the false positive problem during transformation mentioned in the last post, i.e., colonies due to transforamtion of the vector self-ligation without insertion. If I dephosphorylate the vector after double digestion and PCR purification, that should avoid self-ligation. In this situation, the insert should be phosphorylated. Will this solve the false positive problem? Any suggestions? Thanks.