ligation problems - (Dec/13/2005 )

HI,

I want to insert a 2 kb PCR-fragment into the Pgex4T1 vector and transform this to DH5alpha. Both the insert and the vector is cut with EcoRI and XhoI. The insert has restriction sites generated by the PCR primers and the number of adjacent bases should be OK according to the NEB catalgoue.

Protocol:

PCR - 100uL (check aliquot on gel). Clean up with Qiagen PCR Spin columns.
Digest in 50 uL ON at 37 deg C. Digest 5 ug vector in 50 uL. Run all of both on gel, cut bands, extract with QIAquick gel extraction Kit, quantify by OD, and verify by running an aliquot on a gel.

Ligation is 5:1(insert:vector) molar ratio, 50 ng vector in 20 uL. (10 uL 10x buffer, 1 uL T4). 4°C over week-end or 16°C ON

Purification in EtOH

I transform all 10ul of ligation reaction into 50ul of competent cells and plate 50ul of the transformed cells, but still get no colonies.

know the competent cells and all lab reagents are fine and work well, as all my labmates have NO problems using the same reagents.

Any help/advice/comments/thoughts would be greatly appreciated.

Thanks in advance

Lauretta

1st question: you don't do an SOC recovery after transformation? this may give you problems if your ligation is not particularly efficient

2nd question: have you tried different vector/insert ratios?

3rd question: 10ul ligation + 50 ul comp cells seems high to me. for best results, use no more than 5 ul per 100 ul comp cells

4th question: what do you see with your controls? what controls do you perform?

5th question: why do an EtOH ppt after ligation? you can go directly to transformation and save the step.

good luck

I agree with aimikins.. you might be loosing DNA with the ethanol step, and it is important the recovery after transformation.... I use 2 hours in 2 ml LB agitating

hi,
I am new member, please let me know how to begin communication. I have registered but how to send messages, since I have no one in my address book. My interests are similar, for example I am perfoming ligation - transformation experiments to make library.
Your help and time is appreciated.
Vibha.

You can check to see if the ligation worked by loading some of it on a gel, next to your controls (plasmid only, no insert, + ligase) or (plasmid + insert, no ligase). Successful ligations result in the appearance of new bands, and the diminution or loss of the original linear plasmid and linear insert bands. Just remember to save enough of the ligation mix to use to transform your bacteria!

Have you used CIP after vector digestion????

phosphatase should not be necessary, unless the problem is coming from a single-digestion of the vector, which would yield tons of colonies on the ligation-control plate

Lauretta, have you been able to have success with this yet?

[quote name='vibha' date='Dec 18 2005, 04:35 PM' post='34837']
hi,
I am new member, please let me know how to begin communication. I have registered but how to send messages, since I have no one in my address book. My interests are similar, for example I am perfoming ligation - transformation experiments to make library.
Your help and time is appreciated.


hi dear
at first you try different ratios and purify well your pcr product chek at every steps take some controles also during the process of transformation be carefull at the heat shok step and temp at wich you keep at what temp

Hi Laurette,

I agree with Amikins try vector/insert ratios, drop the EtOH precipitation and don't allow your ligation make up more than 10% of your transformation mix.

However, in addition I would blunt end clone (or T/A clone) into another vector and cut out of there. I have found numerous problems trying to follow the number of nucleotides from the end as prescribed by NEB and often add an extra 10 or 15 if possible I think this could very well be the problem.

Hope this helps,

Scott