6 replies to this topic

[litos]

Hi there,

I am having trouble with an apparently straightforward cloning. I need to excise a fragment of approximately 1.7 kb from a 4.8 kb parent vector and join it to a 5.7 kb destination vector. I could not find any proper restriction sites for a cohesive ligation so I had to blunt both the fragment and the destination vector. The enzymes I choose to excise the fragment and open the vector all leave 5' overhangs.

I use NEB's quick blunting kit (T4 polymerase) so I think my fragments are not "polished", but filled (beacause T4 5'->3' polymerase activity). Then, I CIP treat the blunted vector, but not the 1.7 kb fragment, in order to reduce the background in the subsequent ligation. I perform an overnight, 16 ºC, ligation at different molar ratios (1:3, 1:6, 1:10, 1:20) in a final volume of 10 ul. Then, I pick 2 ul of the ligations and transform DH5a competent bacteria, but I dont see any clones. My positive and transformation controls are OK. I repeated the transformation with 4 ul of the ligation, with the same results.

This is annoying me a lot, ¿any idea of what is going wrong? Thanks in advance.

Edited by litos, 18 August 2009 - 12:22 AM.

[stardust]

Hi,

did you purify your backbone after CIP treatment?

Stardust

[litos]

Yes, I perform a restriction digestion on both the backbone and the insert, then heat inactive the reactions. After that, I blunt the fragments, heat inactivate again, CIP the vector and run a gel (I prefer to do only one purification step, beacause of the poor efficiency of the gel extraction kit we use). I think a weak point could be the CIP incubation time or quantity (I use 0.5 unit per microgram of DNA and 1h of incubation, according to the manufacturer's instructions). Maybe I could reduce it, since I heard CIP use to be tricky. Another possible critical step may be in the time of exposure of my DNA to UV light during the gel band excision, but that is only speculation.

Edited by litos, 18 August 2009 - 01:03 AM.

[Clare]

litos, on Aug 18 2009, 09:19 AM, said:

Yes, I perform a restriction digestion on both the backbone and the insert, then heat inactive the reactions. After that, I blunt the fragments, heat inactivate again, CIP the vector and run a gel (I prefer to do only one purification step, beacause of the poor efficiency of the gel extraction kit we use). I think a weak point could be the CIP incubation time or quantity (I use 0.5 unit per microgram of DNA and 1h of incubation, according to the manufacturer's instructions). Maybe I could reduce it, since I heard CIP use to be tricky. Another possible critical step may be in the time of exposure of my DNA to UV light during the gel band excision, but that is only speculation.

You could try adding 2ul of 50% PEG 4000 (for a 20ul ligation) to increase ligation efficiency. Worked for me
Good luck!
Clare

[phage434]

PCR your vector backbone with the restriction enzyme cut sites in the primer 5' ends (don't forget the overhangs).  Purify, RE cut, heat kill, add insert, ligate.  You are not a slave to the set of RE cut sites some bozo (or genius) put into your vector.

[stardust]

Blunt cloning can be tricky. If it doesn't work I do this:

20 µl ligation with 100 ng CIP treated purified vector backbone, 1 µl T4 ligase (NEB), 2 µl ligase buffer, fill up with insert (so I don't add any additional water and I don't stick to defined molar ratios). Incubate 1 h at RT, the ON at 4°C. In the morning add 1 µl ligase again, nothing else, incubate at RT for 2 h, followed by transformation of 1 - 2 µl in electrocompeten cells.

Worked well for me in the past...

BTW, what is the concentration of your insert?

Stardust

[litos]

Thanks to everyone for your kind suggestions.

Stardust, as I said before, I try different vector to insert molar ratios, so I use growing quantities of my insert (being the 1:3 ratio 50 ng plasmid: 34 ng insert). The original concentration of my insert after gel band purification is of about 70 ng/ul.

Litos.