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Ligation/transformation/plates problems - molecular cloning problems (Aug/01/2009 )

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Hello

I am currently trying to ligate a 1.68Kb insert into a 5Kb plasmid but find that when I do a ligation, transform them into competent cells and plate them on agar plates containing ampicillin, I get colonies however, when visualised on a gel after mini-prep there is nothing - the clones do not contain anything! This led to me to think that the initial ligation/transformation hadn't worked and that there was also something wrong with my plates - perhaps not enough ampicillin.

The insert had been digested from another plasmid using EcoRI/HindIII, gel extracted using a Promega kit (visualised under UV) and eluted in water giving a concentration of 4.5ng/ul. The plasmid had also been digested to remove another inserted using the same enzymes and purified in the same way giving 56.9ng/ul. I ran a sample of the plasmid on a gel to make sure it is running at the correct size - it was but I didn't run the insert as I had such little amount of it I didn't want to waste any! I set up ligations using 50ng of plasmid and 16.8ng insert (1:1 ratio), 100ng plasmid and 33.6ng insert (1:1 ratio) and also 50ng plasmid and 50.4ng insert (1:3 ratio) using the ng vector = length insert/length of vector calculation. I then used NEB T4 ligase and its buffer and left at room temperature overnight.

I transformed into ultracompetent cells from Stratagene using their protocol - I am reasonably confident nothing went wrong with this step.

For the plates I made 600ml agar and once it had cooled, added 600ul of 100ug/ml Ampicillin that had been made from a stock of 100mg/ml. Calculations aren't my forte so perhaps I have been diluting the Amp too much without realising? From the 100mg/ml stock I took 1ul and added it to 999ul ddH2O. Is that correct??

I got plenty of colonies and when I chose a few at random to mini-prep, the clones did not contain anything - the gel was completely blank although the DNA ladder was there. The nanodrop also reckoned there was DNA in the samples. As a sort of control I also streaked a glycerol stock of plasmid I had previously made on the plates, this also returned colonies but these actually contained DNA of the correct size.

Any help/advice would be greatly appreciated! I'm very new to cloning and am completely stuck and disheartened at this point!

Thanks

-chantalh-

chantalh on Aug 1 2009, 09:22 AM said:

Hello

I am currently trying to ligate a 1.68Kb insert into a 5Kb plasmid but find that when I do a ligation, transform them into competent cells and plate them on agar plates containing ampicillin, I get colonies however, when visualised on a gel after mini-prep there is nothing - the clones do not contain anything! This led to me to think that the initial ligation/transformation hadn't worked and that there was also something wrong with my plates - perhaps not enough ampicillin.

The insert had been digested from another plasmid using EcoRI/HindIII, gel extracted using a Promega kit (visualised under UV) and eluted in water giving a concentration of 4.5ng/ul. The plasmid had also been digested to remove another inserted using the same enzymes and purified in the same way giving 56.9ng/ul. I ran a sample of the plasmid on a gel to make sure it is running at the correct size - it was but I didn't run the insert as I had such little amount of it I didn't want to waste any! I set up ligations using 50ng of plasmid and 16.8ng insert (1:1 ratio), 100ng plasmid and 33.6ng insert (1:1 ratio) and also 50ng plasmid and 50.4ng insert (1:3 ratio) using the ng vector = length insert/length of vector calculation. I then used NEB T4 ligase and its buffer and left at room temperature overnight.

I transformed into ultracompetent cells from Stratagene using their protocol - I am reasonably confident nothing went wrong with this step.

For the plates I made 600ml agar and once it had cooled, added 600ul of 100ug/ml Ampicillin that had been made from a stock of 100mg/ml. Calculations aren't my forte so perhaps I have been diluting the Amp too much without realising? From the 100mg/ml stock I took 1ul and added it to 999ul ddH2O. Is that correct??

I got plenty of colonies and when I chose a few at random to mini-prep, the clones did not contain anything - the gel was completely blank although the DNA ladder was there. The nanodrop also reckoned there was DNA in the samples. As a sort of control I also streaked a glycerol stock of plasmid I had previously made on the plates, this also returned colonies but these actually contained DNA of the correct size.

Any help/advice would be greatly appreciated! I'm very new to cloning and am completely stuck and disheartened at this point!

Thanks



You're adding 600 ul of 100 ug/ml amp to 600 ml of agar? That's a 1000x dilution, so your final concentration is 0.1 ug/ml or 100 ng/ml. That's way too low. What you need to do is add 600 ul of your "stock" amp to the agar. Put 600 ul of 100 mg/ml into the 600 ml of agar for a final amp concentration of 100 ug/ml.


I hope my lab math is right...

-fishdoc-

fishdoc on Aug 1 2009, 05:40 PM said:

>


You're adding 600 ul of 100 ug/ml amp to 600 ml of agar? That's a 1000x dilution, so your final concentration is 0.1 ug/ml or 100 ng/ml. That's way too low. What you need to do is add 600 ul of your "stock" amp to the agar. Put 600 ul of 100 mg/ml into the 600 ml of agar for a final amp concentration of 100 ug/ml.


I hope my lab math is right...



Ah of course! Thanks a lot for your reply.

I knew it would probably involve my terrible dilution skills! I'll make up some new agar with the 100mg/ml and hopefully it will be problem solved :-)

-chantalh-

I made up new agar using 100mg/ml ampicillin and no colonies with my ligations!

Any ideas on what could be going wrong with the ligation? Is overnight at room temperature too long? Could there be anything in the gel extraction process that would inhibit a ligation?

Any help much appreciated!

-chantalh-

chantalh on Aug 1 2009, 04:22 PM said:

The insert had been digested from another plasmid using EcoRI/HindIII, . I then used NEB T4 ligase and its buffer and left at room temperature overnight.


Thanks



For sticky ends, I would do room temperature ligations for 10-30 minutes, but not over-night. (I do overnight ligation at 16C for blunt ends)
I don't know what can do overnight on your ligation, but maybe this is the reason why your ligation failed ?

-little mouse-

chantalh on Aug 5 2009, 05:55 AM said:

I made up new agar using 100mg/ml ampicillin and no colonies with my ligations!

Any ideas on what could be going wrong with the ligation? Is overnight at room temperature too long? Could there be anything in the gel extraction process that would inhibit a ligation?

Any help much appreciated!




Most of my sticky-end ligations I leave sit overnight at room temp. I don't know of any time that it's caused a problem.

Could be too little DNA in the ligation. Could be a bad insert:vector ratio. Could be not pure enough DNA. Could be too little vector DNA. It could be a lot of things, so I won't go on listing them.

Most of the time when I have a problem with transformations, it's due to starting with not enough vector or insert DNA and having a low concentration in the reaction, having a poor digest, or not cleaning the DNA up well enough after digestion.

I've also had problems where I wasn't using the right E. coli strain to carry the plasmid, or if the copy number of the plasmid is low enough, it doesn't produce enough resistance to grow on that concentration of antibiotics.

-fishdoc-

chantalh on Aug 5 2009, 06:55 PM said:

I made up new agar using 100mg/ml ampicillin and no colonies with my ligations!

Any ideas on what could be going wrong with the ligation? Is overnight at room temperature too long? Could there be anything in the gel extraction process that would inhibit a ligation?

Any help much appreciated!


Can the E.coli with the blank plasmid grow on the media with 100mg/ml ampicillin?

-smallcat227-

Sometimes with cloning you just have to try, try, try again! I do overnight sticky end ligations at rt but sometimes they just don't work. There also might be a lucky spot in your lab that they work better in - for us it was the postdoc's office. It was just a little bit cooler in there. Remember, you only need 1 colony!

-microgirl-

Ok - firstly, with your equations: I find it easy to work out everything using C1V1=C2V2 if I include ALL the zeros. Here is an example
(in grams)
1.0 = 1 gram
0.1 = 100 mg
0.0001 = 100 ug
0.0000001 = 100 ng
and so on

You want: 600 mLs of 100ug/ml
You have stock of 100mg/ml Amp
So C1V1 = C2V2
0.000100 X 0.600 = 0.100 X x
0.00006 = 0.100x
x = 0.0006
x = 600 ul of 100mg/ml stock means you have a final dilution of 100ug/ml. You don't add water to the antibiotic, you use the agar to dilute to the final concentration.

Now, and this is important: Amp degrades really quickly!! Plates kept at 4 degrees C should be discarded after a month. Also, 8 hours at 37 degrees is usually all thats needed to see positive colonies - so if you spread your tfn at 9 am the next day and leave it at 24 hours to the next you are likely to get satellite (negative) colonies - a positive colony grows and degrades all the Amp surrounding it, then suddenly negative, non amp resistant colonies can grow next to the original colony. The solution is to spread your E.coli late in the day, then pull your plates out as soon as you get in and RESTREAK to a fresh plate which you can check later that day.

Hope this helps, A.

-Arqwen-

Thanks everyone for taking the time to reply!

little mouse on Aug 5 2009, 12:03 PM said:

chantalh on Aug 1 2009, 04:22 PM said:

The insert had been digested from another plasmid using EcoRI/HindIII, . I then used NEB T4 ligase and its buffer and left at room temperature overnight.


Thanks



For sticky ends, I would do room temperature ligations for 10-30 minutes, but not over-night. (I do overnight ligation at 16C for blunt ends)
I don't know what can do overnight on your ligation, but maybe this is the reason why your ligation failed ?


I have done a ligation previously at R.T. overnight and didn't have a problem with it - this was using the same plasmid type (although a different aliquot sample to what I'm using now) but with a different insert.

fishdoc on Aug 5 2009, 02:23 PM said:

chantalh on Aug 5 2009, 05:55 AM said:

I made up new agar using 100mg/ml ampicillin and no colonies with my ligations!

Any ideas on what could be going wrong with the ligation? Is overnight at room temperature too long? Could there be anything in the gel extraction process that would inhibit a ligation?

Any help much appreciated!




Most of my sticky-end ligations I leave sit overnight at room temp. I don't know of any time that it's caused a problem.

Could be too little DNA in the ligation. Could be a bad insert:vector ratio. Could be not pure enough DNA. Could be too little vector DNA. It could be a lot of things, so I won't go on listing them.

Most of the time when I have a problem with transformations, it's due to starting with not enough vector or insert DNA and having a low concentration in the reaction, having a poor digest, or not cleaning the DNA up well enough after digestion.

I've also had problems where I wasn't using the right E. coli strain to carry the plasmid, or if the copy number of the plasmid is low enough, it doesn't produce enough resistance to grow on that concentration of antibiotics.


My own feeling would agree that it's probably due to the quality of my DNA following extraction and the concentrations I use in the ligation. I can recover a decent enough concentration of plasmid following extraction but I tend to struggle with my insert concentration after digesting 1ug of it to extract from the pUC plasmid it came in and that has been the limiting factor in my ligations, not being able to vary the ratios as much as I would like due to too little insert DNA.
I think the plasmid I'm using is a low-copy plasmid, it's pAK19 as my insert is the heavy and light chains of an antibody which pAK19 will express as rFab molecules. However I (and others) have been able to grow the plasmid with these competent cells (Stratagene XL10 Golds) on agar plates of that concentration without problem.

smallcat227 on Aug 5 2009, 03:01 PM said:

Can the E.coli with the blank plasmid grow on the media with 100mg/ml ampicillin?


Actually I haven't tried this! I was running low on bugs at the time so didn't want to 'waste' any! Although this would have been a useful control - we are ordering new stocks of everything, from bugs to buffers and enzymes so I will try this out then and see if any colonies appear.

microgirl on Aug 5 2009, 05:22 PM said:

Sometimes with cloning you just have to try, try, try again! I do overnight sticky end ligations at rt but sometimes they just don't work. There also might be a lucky spot in your lab that they work better in - for us it was the postdoc's office. It was just a little bit cooler in there. Remember, you only need 1 colony!


I'm definitely learning this the hard way! Maybe I will try setting some up on my bench at R.T. and others in a 16 degree water bath in the cold room and perhaps another near the air-conditioning in the lab! At this point I'll give anything a go!

Arqwen on Aug 6 2009, 11:07 AM said:

Ok - firstly, with your equations: I find it easy to work out everything using C1V1=C2V2 if I include ALL the zeros. Here is an example
(in grams)
1.0 = 1 gram
0.1 = 100 mg
0.0001 = 100 ug
0.0000001 = 100 ng
and so on

You want: 600 mLs of 100ug/ml
You have stock of 100mg/ml Amp
So C1V1 = C2V2
0.000100 X 0.600 = 0.100 X x
0.00006 = 0.100x
x = 0.0006
x = 600 ul of 100mg/ml stock means you have a final dilution of 100ug/ml. You don't add water to the antibiotic, you use the agar to dilute to the final concentration.

Now, and this is important: Amp degrades really quickly!! Plates kept at 4 degrees C should be discarded after a month. Also, 8 hours at 37 degrees is usually all thats needed to see positive colonies - so if you spread your tfn at 9 am the next day and leave it at 24 hours to the next you are likely to get satellite (negative) colonies - a positive colony grows and degrades all the Amp surrounding it, then suddenly negative, non amp resistant colonies can grow next to the original colony. The solution is to spread your E.coli late in the day, then pull your plates out as soon as you get in and RESTREAK to a fresh plate which you can check later that day.

Hope this helps, A.

Thanks for the calculations, I vaguely remember the equation from my school Chemistry years ago!
I try to only leave the plates for 17-20hrs, as recommended in the Stratagene protocol of my cells but I will certainly give the restreaking a go.


Thanks again everyone, hopefully your suggestions will work as this is the very basis of my PhD - I can't go any further until this insert is cloned into the vector!

-chantalh-
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