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Help: try ligation but see nothing from the gels


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9 replies to this topic

#1 netnus

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Posted 22 September 2004 - 12:08 PM

I recently want to ligate the DNA insert (500bp) to the vector plasmid pET-9a. They have been double-digested using the same restriction enzymes(Bamh1 & Nde1). I bought the T4 ligase from Fermentas. They give me the protocol as following:
1.Prepare 5-10ul (50-400ng) vector DNA and foreign DNA to be inserted;
2. Add
10x ligation buffer 2ul
deionized water to 20ul;
T4 DNA ligase 1-2unit (for sticky ends)
Vortex the tube and spin down for 3-5 sec
3. incubate the mixture for 1 hour at 22 degree;
4. inactivate T4 ligase by heating the rxn mixture at 65 degree for 10 min.

While I just follow the protocol, I get nothing. I repeat the exp for several times but still get nothing. Eventually I try using the mixture for transformation, but still get nothing growing on the LB-agar plate.
I am really upset since I run out of the DNA insert and Vector Plasmid but I get nothing.
I really hope someone can help me out. It seems like the insert and the vector plasmid are completely degraded to small fragements.
Did anyone have the same problem before? How did you solve it?
Greatly appreciate your suggestions !

netnus

#2 labrat

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Posted 22 September 2004 - 04:14 PM

For cloning, always include controls both positive and negative so that you could track back what is wrong. A few suggestions:

How was your insert prepared, are you sure both enzyme cut into your insert?
After digesting your plasmid, did you check if your plasmid has been linerized? Even sometimes the vendor says that the two enzymes are compatible in one buffer, they may actually not.
Do a positive control using some uncut plasmid but is antibiotics resistant to see the transformation is OK and your compotent cells are OK.
Aliquot your ligation buffer and store it at -20C, don't think it is just regular buffer. It contains ATP which may degradate after multiple thawings.

#3 kant0008

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Posted 22 September 2004 - 04:21 PM

Hi,
do you know your plasmid and insert concentrations before you start? You shoudl be able to see at least something, even if you get degradation.
Suggestions:
Don't vortex, pipetting up and down a few times should do the trick.
Don't overheat your mix. 65 should be ok for DNA, but don't leave it too long.
I do my ligations overnight always, at 4C, I don't believe them when they say 1hr is enough :huh:
I agree with labrat, have positive controls so that you know exactly what step is failing. And do check complete digestion too.
Good luck

#4 netnus

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Posted 23 September 2004 - 10:38 AM

For cloning, always include controls both positive and negative so that you could track back what is wrong. A few suggestions:

How was your insert prepared, are you sure both enzyme cut into your insert?
After digesting your plasmid, did you check if your plasmid has been linerized? Even sometimes the vendor says that the two enzymes are compatible in one buffer, they may actually not.
Do a positive control using some uncut plasmid but is antibiotics resistant to see the transformation is OK and your compotent cells are OK.
Aliquot your ligation buffer and store it at -20C, don't think it is just regular buffer. It contains ATP which may degradate after multiple thawings.

Thanks for your two's answers!
Since I am a newbie in Molecular biology,could you explain me how I could do the positive and negative controls?
I check the digested insert and plasmid by runing the gels to compare them with the undigested inset and plasmid. And I can obviously see the different location in the gels. So I suppose they have been cut. Do you think they are not cut by the enzyme? How can I control them?
Greatly appreciate your answers again!

#5 kant0008

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Posted 23 September 2004 - 09:10 PM

Hi again,
you say you see a difference in fragment sizes cut vs. uncut even with insert? Are you cutting it out of another vector?

OK, about controls.
Ligation positive control: Cut a vector with one enzyme only(so that it is able to reclose), and religate. This should give you a closed plasmid (and colonies if you transform) to make sure ligase is working
Ligation/Transformation negative control: Double digest your vector, purify as normal. Religate vector- if your digest works well then this ligation should Not happen, and you should get zero colonies on transformation.
Transformation positive control: trasform uncut plasmid, should have lots of colonies.

#6 netnus

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Posted 24 September 2004 - 07:08 AM

Hi again,
you say you see a difference in fragment sizes cut vs. uncut even with insert? Are you cutting it out of another vector?

OK, about controls.
Ligation positive control: Cut a vector with one enzyme only(so that it is able to reclose), and religate. This should give you a closed plasmid (and colonies if you transform) to make sure ligase is working
Ligation/Transformation negative control: Double digest your vector, purify as normal. Religate vector- if your digest works well then this ligation should Not happen, and you should get zero colonies on transformation.
Transformation positive control: trasform uncut plasmid, should have lots of colonies.

Yes! I do sequential-digest the insert and the plasmid vector. The BamH1 doesn't cut very efficiently, but I still can extract the digested vector from the gel (my colleagues doubt I cut the wrong band from the gel since I cut the band with stongest intensity,maybe it is not the cut vector?)
I will try the positive and negative control later!
Thanks a lot!

#7 kant0008

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Posted 26 September 2004 - 04:12 PM

Hi,
just thought I'd mention that to get the cut vector you can go directly by size, as the cut vector will migrate at exactly the molecular size it is, so go by marker, not by intensity

#8 vetticus3

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Posted 28 September 2004 - 07:04 PM

Hey,
The research officer in my lab refuses to use Fermentas T4 ligase and buffer. She says that they're useless, and when our lab ran out of another brand, and she had to use Fermentas, her plates were whacked. There were colonies on the controls, and nothing on the experiment.
When we finally got her favourite brand back, everything worked.
Maybe beg/borrow/steal someone elses ligase.
Vetticus

#9 netnus

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Posted 29 September 2004 - 01:20 PM

Hey,
The research officer in my lab refuses to use Fermentas T4 ligase and buffer.  She says that they're useless, and when our lab ran out of another brand, and she had to use Fermentas, her plates were whacked.  There were colonies on the controls, and nothing on the experiment. 
When we finally got her favourite brand back, everything worked.
Maybe beg/borrow/steal someone elses ligase.
Vetticus

Your research officer is really funny.
Except T4 ligase, what else ligase do you use? Do you means we'd better use T4 ligase from other companies? Which company would you like to recomend?

Thanks!

#10 spn25

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Posted 29 September 2004 - 03:22 PM

did you sap your vector after the digestion? you should do that.
sap for 1 hr at 37 and 20min at 65 degree.
I normally carry out the ligation with 16 degree for 16hrs/overnite.
my recipe is:
05ul( or 1ul or 1.5ul) of insert
1ul of vector
1ul of ligase
1ul of ligase buffer
and top up to 10 ul with ddh20.

also do a negative control without any insert to see your vector is fully cut.




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