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ligation problem


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6 replies to this topic

#1 wizzkid

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Posted 07 March 2010 - 03:10 AM

Hi,

I performed a ligation reaction using the insert:vector molar ratios 3:1, 1:1 and 6:1. While i did achieve colonies on the 3:1 plate, the 1:1 and 6:1 had absolutely no colonies. why is this? i performed suitable ligation controls and they were all ok,
would appreciate some help with this :wacko: thanks

#2 Denny

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Posted 07 March 2010 - 03:22 PM

Hi,

I performed a ligation reaction using the insert:vector molar ratios 3:1, 1:1 and 6:1. While i did achieve colonies on the 3:1 plate, the 1:1 and 6:1 had absolutely no colonies. why is this? i performed suitable ligation controls and they were all ok,
would appreciate some help with this :wacko: thanks


There a many factors to consider when making ligations, a few questions first:
What controls did you use?
What ligase are you using?
What is the size of vector and insert?
Are your ends sticky or blunt?
Are you actually calculating the molar ratios?

The reason for using different molar ratios is that some work and some don't :P

#3 wizzkid

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Posted 08 March 2010 - 01:08 AM

Hi,

I performed a ligation reaction using the insert:vector molar ratios 3:1, 1:1 and 6:1. While i did achieve colonies on the 3:1 plate, the 1:1 and 6:1 had absolutely no colonies. why is this? i performed suitable ligation controls and they were all ok,
would appreciate some help with this :) thanks


There a many factors to consider when making ligations, a few questions first:
What controls did you use?
What ligase are you using?
What is the size of vector and insert?
Are your ends sticky or blunt?
Are you actually calculating the molar ratios?

The reason for using different molar ratios is that some work and some don't :D




hey thanks for the reply. to answer your questions:
my controls were -positive control where it was just my vector without insert that was not alkaline phosphatase treated
-negative control was just my vector but this time it was alkaline phosphatase treated

the ligase i used was T4 dna ligase (NEB) and used the appropiate 10X ligase buffer (NEB)
the size of my vector is 6Kb and my insert is 600bp and they were cut with a restriction enzyme to give sticky ends
and yes i am using the insert:vector calculation= [conc of vector]Xbp size of insert/bp size of vector X ratio insert:vector
i did have trouble getting the conc of my vector (im a beginner) using a spec, so i ran a gel and viewed the intensity diff on the gel between the insert and vector. i then proceeded to make up my ligations as follows, say i took 6 microliters of vector i would add 2.5 microliters insert.

#4 phage434

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Posted 08 March 2010 - 05:26 AM

Why do you need more than one colony that is correct?

#5 wizzkid

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Posted 08 March 2010 - 05:58 AM

Why do you need more than one colony that is correct?


Hi phage342,
yes i want a few colonies, just in case my insert is in the wrong orientation in some. thats what my supervisor told me to do.

#6 Denny

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Posted 08 March 2010 - 08:25 AM

Hi,

I performed a ligation reaction using the insert:vector molar ratios 3:1, 1:1 and 6:1. While i did achieve colonies on the 3:1 plate, the 1:1 and 6:1 had absolutely no colonies. why is this? i performed suitable ligation controls and they were all ok,
would appreciate some help with this :D thanks


There a many factors to consider when making ligations, a few questions first:
What controls did you use?
What ligase are you using?
What is the size of vector and insert?
Are your ends sticky or blunt?
Are you actually calculating the molar ratios?

The reason for using different molar ratios is that some work and some don't :)




hey thanks for the reply. to answer your questions:
my controls were -positive control where it was just my vector without insert that was not alkaline phosphatase treated
-negative control was just my vector but this time it was alkaline phosphatase treated

the ligase i used was T4 dna ligase (NEB) and used the appropiate 10X ligase buffer (NEB)
the size of my vector is 6Kb and my insert is 600bp and they were cut with a restriction enzyme to give sticky ends
and yes i am using the insert:vector calculation= [conc of vector]Xbp size of insert/bp size of vector X ratio insert:vector
i did have trouble getting the conc of my vector (im a beginner) using a spec, so i ran a gel and viewed the intensity diff on the gel between the insert and vector. i then proceeded to make up my ligations as follows, say i took 6 microliters of vector i would add 2.5 microliters insert.


As phage said, it only takes one colony and you're off and running.

Let's look at your controls a second and interpret what they are telling you.

With the assumption that you are running your insert and vector on a gel after restriction digest (if you don't you will get a small percentage of uncut vector and won't know if your restriction digest worked and may be starting with all circular vector not to mention extra enzymes and reagents in your ligation reaction that will cause problems):

1) "positive control" - vector untreated with phosphatase
- if you get colonies it means your vector is re-ligating. This tells you that your ligase is working and that your cells are competent.
- if you don't get colonies, either your ligase is not working and your vector remains linear or your cells are not competent

2) "negative control" - vector treated with phosphatase
- if you get colonies the ligase is working, your cells are competent but the phosphatase treatment is either not working or incubation time was too short.
- if you don't get colonies - the phosphatase is working and your cells are competent

The controls won't tell you anything about your molar ratios. :)

Eyeballing band intensity is okay as long as you're not having ligation problems. Ask your lab mates for help in using the spec and figuring out your concentrations so you will know how to do it in the future! :)

#7 wizzkid

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Posted 08 March 2010 - 11:46 AM

Hi,

I performed a ligation reaction using the insert:vector molar ratios 3:1, 1:1 and 6:1. While i did achieve colonies on the 3:1 plate, the 1:1 and 6:1 had absolutely no colonies. why is this? i performed suitable ligation controls and they were all ok,
would appreciate some help with this :D thanks


There a many factors to consider when making ligations, a few questions first:
What controls did you use?
What ligase are you using?
What is the size of vector and insert?
Are your ends sticky or blunt?
Are you actually calculating the molar ratios?

The reason for using different molar ratios is that some work and some don't :lol:




hey thanks for the reply. to answer your questions:
my controls were -positive control where it was just my vector without insert that was not alkaline phosphatase treated
-negative control was just my vector but this time it was alkaline phosphatase treated

the ligase i used was T4 dna ligase (NEB) and used the appropiate 10X ligase buffer (NEB)
the size of my vector is 6Kb and my insert is 600bp and they were cut with a restriction enzyme to give sticky ends
and yes i am using the insert:vector calculation= [conc of vector]Xbp size of insert/bp size of vector X ratio insert:vector
i did have trouble getting the conc of my vector (im a beginner) using a spec, so i ran a gel and viewed the intensity diff on the gel between the insert and vector. i then proceeded to make up my ligations as follows, say i took 6 microliters of vector i would add 2.5 microliters insert.


As phage said, it only takes one colony and you're off and running.

Let's look at your controls a second and interpret what they are telling you.

With the assumption that you are running your insert and vector on a gel after restriction digest (if you don't you will get a small percentage of uncut vector and won't know if your restriction digest worked and may be starting with all circular vector not to mention extra enzymes and reagents in your ligation reaction that will cause problems):

1) "positive control" - vector untreated with phosphatase
- if you get colonies it means your vector is re-ligating. This tells you that your ligase is working and that your cells are competent.
- if you don't get colonies, either your ligase is not working and your vector remains linear or your cells are not competent

2) "negative control" - vector treated with phosphatase
- if you get colonies the ligase is working, your cells are competent but the phosphatase treatment is either not working or incubation time was too short.
- if you don't get colonies - the phosphatase is working and your cells are competent

The controls won't tell you anything about your molar ratios. :P

Eyeballing band intensity is okay as long as you're not having ligation problems. Ask your lab mates for help in using the spec and figuring out your concentrations so you will know how to do it in the future! :D



cheers denny for the help ans effort to ans my question :D




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