Ligation/transformation/plates problems - molecular cloning problems (Aug/01/2009 )
Hi - me again!
I'm afraid that my ligations still aren't working!
I started everything again, ordered all new reagents such as competent bugs, restriction enzymes, ligases etc and still not a single clone on my plates! 
I'm pretty sure the problem is in the gel extraction and/or ligation steps.
I'd appreciate any advice on the following questions (I don't have anyone in the lab to ask, unfortunately):
- in making the gel could there be any factors involving the TAE used that might inhibit the ligation e.g. any contaminants in the TAE, how accurately it was made from a more concentrated stock etc?
- how long is too long to keep the DNA exposed to UV? I try to minimise the exposure time as much as possible but perhaps I'm just not working quickly enough
- fishdoc mentioned about too low concentration of vector and insert in the ligation reaction - could you be more specific on how is too low? I tend to use either 50ng or 100ng of vector and then use a calculation to work out th einsert based on this vector amount
- Before gel extraction the plasmid and insert had been digested at 37 degrees overnight - is this too long a digestion? Is that likely to interfere/affect later results?
Thanks again guys!
-chantalh-
chantalh on Sep 18 2009, 07:36 AM said:
Hi - me again!
I'm afraid that my ligations still aren't working!
I started everything again, ordered all new reagents such as competent bugs, restriction enzymes, ligases etc and still not a single clone on my plates!
I'm pretty sure the problem is in the gel extraction and/or ligation steps.
I'd appreciate any advice on the following questions (I don't have anyone in the lab to ask, unfortunately):
- in making the gel could there be any factors involving the TAE used that might inhibit the ligation e.g. any contaminants in the TAE, how accurately it was made from a more concentrated stock etc?
- how long is too long to keep the DNA exposed to UV? I try to minimise the exposure time as much as possible but perhaps I'm just not working quickly enough
- fishdoc mentioned about too low concentration of vector and insert in the ligation reaction - could you be more specific on how is too low? I tend to use either 50ng or 100ng of vector and then use a calculation to work out th einsert based on this vector amount
- Before gel extraction the plasmid and insert had been digested at 37 degrees overnight - is this too long a digestion? Is that likely to interfere/affect later results?
Thanks again guys!
I'm afraid that my ligations still aren't working!
I started everything again, ordered all new reagents such as competent bugs, restriction enzymes, ligases etc and still not a single clone on my plates!
I'm pretty sure the problem is in the gel extraction and/or ligation steps.
I'd appreciate any advice on the following questions (I don't have anyone in the lab to ask, unfortunately):
- in making the gel could there be any factors involving the TAE used that might inhibit the ligation e.g. any contaminants in the TAE, how accurately it was made from a more concentrated stock etc?
- how long is too long to keep the DNA exposed to UV? I try to minimise the exposure time as much as possible but perhaps I'm just not working quickly enough
- fishdoc mentioned about too low concentration of vector and insert in the ligation reaction - could you be more specific on how is too low? I tend to use either 50ng or 100ng of vector and then use a calculation to work out th einsert based on this vector amount
- Before gel extraction the plasmid and insert had been digested at 37 degrees overnight - is this too long a digestion? Is that likely to interfere/affect later results?
Thanks again guys!
If you are following the gel extraction procedure properly, there should be no worries with the TAE. As long as you're getting a concentration of DNA following purification that has a good 260/280 ratio, there shouldn't be an issue.
Even if you exposed the DNA to the UV too long, you should still get an insert. The UV damage (if any) would manifest itself as mutations, but it shouldn't inhibit ligation or transformation.
Depending on what ligase you're using, your optimal DNA concentration will vary. I use T4 DNA ligase, and it recommends between 1 and 10 uM of DNA in the concentration. However, someone I work with swears the concentration should be higher than 30 uM based on Maniatis. Honestly, I hardly ever get down to less than 10 uM per reaction, and my ligations typically work just fine. I've also frequently gone against the instruction to only add 2 or 5 ul or so of the ligation to the competent cells. I've added 10-30 ul a number of times with successful results. The point is, you can try many conditions, and they may not be optimal, but for the most part, they should still work to some degree.
Overnight digestion usually is OK in my experience, but if you're not digesting under optimal conditions, there can be star activity, and that could reduce how well everything works.
I recently went through a battle with ligations that wouldn't work. Turned out it was because I was using vector that had been digested a few weeks before use, stored in water, and kept at -20. The vector must've degraded slightly, because I would get ZERO colonies. When I repeated with freshly digested vector, everything was good.
Is it possible the gene you're trying to clone is toxic to E. coli?
Have you tried transforming the empty vector into your E. coli strain to see if that works?
A way to test whether or not your ligation is working is to PCR amplify using the ligation reaction as a template. Use primers in the vector that would flank the insert. If you get the correct sized band in the reaction, the ligation was successful, at least to a degree in which you can amplify the expected product.
-fishdoc-
fishdoc on Sep 18 2009, 02:37 PM said:
If you are following the gel extraction procedure properly, there should be no worries with the TAE. As long as you're getting a concentration of DNA following purification that has a good 260/280 ratio, there shouldn't be an issue.
Even if you exposed the DNA to the UV too long, you should still get an insert. The UV damage (if any) would manifest itself as mutations, but it shouldn't inhibit ligation or transformation.
Depending on what ligase you're using, your optimal DNA concentration will vary. I use T4 DNA ligase, and it recommends between 1 and 10 uM of DNA in the concentration. However, someone I work with swears the concentration should be higher than 30 uM based on Maniatis. Honestly, I hardly ever get down to less than 10 uM per reaction, and my ligations typically work just fine. I've also frequently gone against the instruction to only add 2 or 5 ul or so of the ligation to the competent cells. I've added 10-30 ul a number of times with successful results. The point is, you can try many conditions, and they may not be optimal, but for the most part, they should still work to some degree.
Overnight digestion usually is OK in my experience, but if you're not digesting under optimal conditions, there can be star activity, and that could reduce how well everything works.
I recently went through a battle with ligations that wouldn't work. Turned out it was because I was using vector that had been digested a few weeks before use, stored in water, and kept at -20. The vector must've degraded slightly, because I would get ZERO colonies. When I repeated with freshly digested vector, everything was good.
Is it possible the gene you're trying to clone is toxic to E. coli?
Have you tried transforming the empty vector into your E. coli strain to see if that works?
A way to test whether or not your ligation is working is to PCR amplify using the ligation reaction as a template. Use primers in the vector that would flank the insert. If you get the correct sized band in the reaction, the ligation was successful, at least to a degree in which you can amplify the expected product.
Even if you exposed the DNA to the UV too long, you should still get an insert. The UV damage (if any) would manifest itself as mutations, but it shouldn't inhibit ligation or transformation.
Depending on what ligase you're using, your optimal DNA concentration will vary. I use T4 DNA ligase, and it recommends between 1 and 10 uM of DNA in the concentration. However, someone I work with swears the concentration should be higher than 30 uM based on Maniatis. Honestly, I hardly ever get down to less than 10 uM per reaction, and my ligations typically work just fine. I've also frequently gone against the instruction to only add 2 or 5 ul or so of the ligation to the competent cells. I've added 10-30 ul a number of times with successful results. The point is, you can try many conditions, and they may not be optimal, but for the most part, they should still work to some degree.
Overnight digestion usually is OK in my experience, but if you're not digesting under optimal conditions, there can be star activity, and that could reduce how well everything works.
I recently went through a battle with ligations that wouldn't work. Turned out it was because I was using vector that had been digested a few weeks before use, stored in water, and kept at -20. The vector must've degraded slightly, because I would get ZERO colonies. When I repeated with freshly digested vector, everything was good.
Is it possible the gene you're trying to clone is toxic to E. coli?
Have you tried transforming the empty vector into your E. coli strain to see if that works?
A way to test whether or not your ligation is working is to PCR amplify using the ligation reaction as a template. Use primers in the vector that would flank the insert. If you get the correct sized band in the reaction, the ligation was successful, at least to a degree in which you can amplify the expected product.
Thanks for the speedy reply.
I'm also using T4 ligase, from NEB.
To be honest I've never even looked at the concentration of DNA used in uM, I use the Promega online BioMAth calculator and hope that has done the work for me!
The vector is freshy digested in that I digest it overnight then the following day extract and set up the ligation.
I don't believe the gene is toxic to E.Coli, it is an immunoglobulin heavy and light chain.
I have tried transforming undigested vector and that works fine, I get plenty of colonies containing the plasmid+insert. I want to use this plasmid to ligate to another insert but I wanted to check that the extracted plasmid would ligate to an insert etc so as a control what I'm doing at the moment is actually trying to ligate the insert and the plasmid that I digested back together before I use the plasmid for my intended insert ligation (hope that makes sense!).
Someone in another lab has recommended shortening my restriction digest time to 1hr and then heat inactivation at 65 degrees for 20mins followed by the Fermentas Rapid Ligation kit for 10mins at R.T. so I'll give that and your PCR advice a go! If that doesn't work I quit!
-chantalh-
chantalh on Sep 18 2009, 09:19 AM said:
fishdoc on Sep 18 2009, 02:37 PM said:
If you are following the gel extraction procedure properly, there should be no worries with the TAE. As long as you're getting a concentration of DNA following purification that has a good 260/280 ratio, there shouldn't be an issue.
Even if you exposed the DNA to the UV too long, you should still get an insert. The UV damage (if any) would manifest itself as mutations, but it shouldn't inhibit ligation or transformation.
Depending on what ligase you're using, your optimal DNA concentration will vary. I use T4 DNA ligase, and it recommends between 1 and 10 uM of DNA in the concentration. However, someone I work with swears the concentration should be higher than 30 uM based on Maniatis. Honestly, I hardly ever get down to less than 10 uM per reaction, and my ligations typically work just fine. I've also frequently gone against the instruction to only add 2 or 5 ul or so of the ligation to the competent cells. I've added 10-30 ul a number of times with successful results. The point is, you can try many conditions, and they may not be optimal, but for the most part, they should still work to some degree.
Overnight digestion usually is OK in my experience, but if you're not digesting under optimal conditions, there can be star activity, and that could reduce how well everything works.
I recently went through a battle with ligations that wouldn't work. Turned out it was because I was using vector that had been digested a few weeks before use, stored in water, and kept at -20. The vector must've degraded slightly, because I would get ZERO colonies. When I repeated with freshly digested vector, everything was good.
Is it possible the gene you're trying to clone is toxic to E. coli?
Have you tried transforming the empty vector into your E. coli strain to see if that works?
A way to test whether or not your ligation is working is to PCR amplify using the ligation reaction as a template. Use primers in the vector that would flank the insert. If you get the correct sized band in the reaction, the ligation was successful, at least to a degree in which you can amplify the expected product.
Even if you exposed the DNA to the UV too long, you should still get an insert. The UV damage (if any) would manifest itself as mutations, but it shouldn't inhibit ligation or transformation.
Depending on what ligase you're using, your optimal DNA concentration will vary. I use T4 DNA ligase, and it recommends between 1 and 10 uM of DNA in the concentration. However, someone I work with swears the concentration should be higher than 30 uM based on Maniatis. Honestly, I hardly ever get down to less than 10 uM per reaction, and my ligations typically work just fine. I've also frequently gone against the instruction to only add 2 or 5 ul or so of the ligation to the competent cells. I've added 10-30 ul a number of times with successful results. The point is, you can try many conditions, and they may not be optimal, but for the most part, they should still work to some degree.
Overnight digestion usually is OK in my experience, but if you're not digesting under optimal conditions, there can be star activity, and that could reduce how well everything works.
I recently went through a battle with ligations that wouldn't work. Turned out it was because I was using vector that had been digested a few weeks before use, stored in water, and kept at -20. The vector must've degraded slightly, because I would get ZERO colonies. When I repeated with freshly digested vector, everything was good.
Is it possible the gene you're trying to clone is toxic to E. coli?
Have you tried transforming the empty vector into your E. coli strain to see if that works?
A way to test whether or not your ligation is working is to PCR amplify using the ligation reaction as a template. Use primers in the vector that would flank the insert. If you get the correct sized band in the reaction, the ligation was successful, at least to a degree in which you can amplify the expected product.
Thanks for the speedy reply.
I'm also using T4 ligase, from NEB.
To be honest I've never even looked at the concentration of DNA used in uM, I use the Promega online BioMAth calculator and hope that has done the work for me!
The vector is freshy digested in that I digest it overnight then the following day extract and set up the ligation.
I don't believe the gene is toxic to E.Coli, it is an immunoglobulin heavy and light chain.
I have tried transforming undigested vector and that works fine, I get plenty of colonies containing the plasmid+insert. I want to use this plasmid to ligate to another insert but I wanted to check that the extracted plasmid would ligate to an insert etc so as a control what I'm doing at the moment is actually trying to ligate the insert and the plasmid that I digested back together before I use the plasmid for my intended insert ligation (hope that makes sense!).
Someone in another lab has recommended shortening my restriction digest time to 1hr and then heat inactivation at 65 degrees for 20mins followed by the Fermentas Rapid Ligation kit for 10mins at R.T. so I'll give that and your PCR advice a go! If that doesn't work I quit!
First off, I need to amend my comment... it's not 1-10 uM, it's 1-10 ug/ml (I think). Here is a FAQ regarding T4 DNA ligase: http://www.neb.com/nebecomm/products/faqproductM0202.asp#346
After you digest your vector and your insert, do you gel purify or reaction/PCR purify your product? If you do not gel purify, have you checked the quality of your purified digested DNA? We had a vector one time that claimed KpnI was a single cutter, but it actually cut twice, and removed part of a selection gene (it was not a commercially produced plasmid).
Edit: nevermind, I see you are gel purifying. Are you still using the prep of the insert DNA that is 4.5 ng/ul? If so, you may be able to amplify that piece of DNA by PCR rather than digesting it, and be able to get a higher concentration to work with.
-fishdoc-
chantalh on Sep 18 2009, 10:19 AM said:
fishdoc on Sep 18 2009, 02:37 PM said:
If you are following the gel extraction procedure properly, there should be no worries with the TAE. As long as you're getting a concentration of DNA following purification that has a good 260/280 ratio, there shouldn't be an issue.
Even if you exposed the DNA to the UV too long, you should still get an insert. The UV damage (if any) would manifest itself as mutations, but it shouldn't inhibit ligation or transformation.
Depending on what ligase you're using, your optimal DNA concentration will vary. I use T4 DNA ligase, and it recommends between 1 and 10 uM of DNA in the concentration. However, someone I work with swears the concentration should be higher than 30 uM based on Maniatis. Honestly, I hardly ever get down to less than 10 uM per reaction, and my ligations typically work just fine. I've also frequently gone against the instruction to only add 2 or 5 ul or so of the ligation to the competent cells. I've added 10-30 ul a number of times with successful results. The point is, you can try many conditions, and they may not be optimal, but for the most part, they should still work to some degree.
Overnight digestion usually is OK in my experience, but if you're not digesting under optimal conditions, there can be star activity, and that could reduce how well everything works.
I recently went through a battle with ligations that wouldn't work. Turned out it was because I was using vector that had been digested a few weeks before use, stored in water, and kept at -20. The vector must've degraded slightly, because I would get ZERO colonies. When I repeated with freshly digested vector, everything was good.
Is it possible the gene you're trying to clone is toxic to E. coli?
Have you tried transforming the empty vector into your E. coli strain to see if that works?
A way to test whether or not your ligation is working is to PCR amplify using the ligation reaction as a template. Use primers in the vector that would flank the insert. If you get the correct sized band in the reaction, the ligation was successful, at least to a degree in which you can amplify the expected product.
Even if you exposed the DNA to the UV too long, you should still get an insert. The UV damage (if any) would manifest itself as mutations, but it shouldn't inhibit ligation or transformation.
Depending on what ligase you're using, your optimal DNA concentration will vary. I use T4 DNA ligase, and it recommends between 1 and 10 uM of DNA in the concentration. However, someone I work with swears the concentration should be higher than 30 uM based on Maniatis. Honestly, I hardly ever get down to less than 10 uM per reaction, and my ligations typically work just fine. I've also frequently gone against the instruction to only add 2 or 5 ul or so of the ligation to the competent cells. I've added 10-30 ul a number of times with successful results. The point is, you can try many conditions, and they may not be optimal, but for the most part, they should still work to some degree.
Overnight digestion usually is OK in my experience, but if you're not digesting under optimal conditions, there can be star activity, and that could reduce how well everything works.
I recently went through a battle with ligations that wouldn't work. Turned out it was because I was using vector that had been digested a few weeks before use, stored in water, and kept at -20. The vector must've degraded slightly, because I would get ZERO colonies. When I repeated with freshly digested vector, everything was good.
Is it possible the gene you're trying to clone is toxic to E. coli?
Have you tried transforming the empty vector into your E. coli strain to see if that works?
A way to test whether or not your ligation is working is to PCR amplify using the ligation reaction as a template. Use primers in the vector that would flank the insert. If you get the correct sized band in the reaction, the ligation was successful, at least to a degree in which you can amplify the expected product.
Thanks for the speedy reply.
I'm also using T4 ligase, from NEB.
To be honest I've never even looked at the concentration of DNA used in uM, I use the Promega online BioMAth calculator and hope that has done the work for me!
The vector is freshy digested in that I digest it overnight then the following day extract and set up the ligation.
I don't believe the gene is toxic to E.Coli, it is an immunoglobulin heavy and light chain.
I have tried transforming undigested vector and that works fine, I get plenty of colonies containing the plasmid+insert. I want to use this plasmid to ligate to another insert but I wanted to check that the extracted plasmid would ligate to an insert etc so as a control what I'm doing at the moment is actually trying to ligate the insert and the plasmid that I digested back together before I use the plasmid for my intended insert ligation (hope that makes sense!).
Someone in another lab has recommended shortening my restriction digest time to 1hr and then heat inactivation at 65 degrees for 20mins followed by the Fermentas Rapid Ligation kit for 10mins at R.T. so I'll give that and your PCR advice a go! If that doesn't work I quit!
with EcoRI I would definitely not digest more than a couple of hours, this very well could be the source of your problem....however I would do things as you did before (ie gel purify) just don't digest so long!!! One thing I will say, is that this type of ligation SHOULD be so simple, that if you are getting NOTHING, something if fundamentally wrong. I think to many people play with ligation conditions when it is something else wrong, ie, two different sticky ends on both the plasmid and insert will result in the correct clone and successful transformation under a huge variety of ligation conditions, so their is something major that is wrong -- DNA degraded (star activity), no ATP in ligation (you can run the ligation on a gel), some major inhibitor, etc etc, I really doubt its a concentration problem. Good luck!! Warren..
-Warren-