9 replies to this topic

[sktewarybt1993]

Hi,

I am doing a ligation of 1.7 kb insert with the 7.7 kb vector. After ligation i get no colony. When i run the ligation product i see the 7.7 kb vector make a concatamer of 15 kb and 1.7 kb insert make a 3.4 kb cocatamer. but i see no supercoil. My 260/280 ratio is arounf 1.76 for both the DNA. I tried 1:3, 1:2,1:1 vector:insert ratio, also i tried the vector:insert ratio of 3:1 and 6:1 but still no colony. My plates are Ok, cells are Ok i have confirmed it by doing the control side by side. The control work weel

Please give me some suggestion if anyone have find such a problem with the ligation and have successfully overcome such problem.

Regards
sunil

[phage434]

How are your DNA fragments made?  Cut?  Purified?  How much are you using?  How are you ligating? (temp, enzyme).  We need to know a lot more to debug this.  What controls were run?

[GMoorthy]

phage434, on Apr 13 2009, 05:20 PM, said:

How are your DNA fragments made?  Cut?  Purified?  How much are you using?  How are you ligating? (temp, enzyme).  We need to know a lot more to debug this.  What controls were run?

Hi, I am having a similar problem.  My vector is around 4.5 kb but after the ligation it runs at >10 kb, suggesting that it forms concatamers.  In the presence of the insert, there is a smear at > 10 kb suggesting some annealing between the vector and the insert is taking place but the vector also seems to be self-ligating. No colonies emerged after transformation.

The vector and insert was PCRed, then gel-extracted and double digested with Nhe1 and Spe1 (15 units of each enzyme for 600 ng DNA) at 37 degrees for 2 hrs. Then both were purified.  Vector was CIPed (0.3 units for 600 ng) in NEB buffer 3 at 37 degrees for 1 hour.  It was purified again before ligation (50 ng vector +/- 250 ng insert) in a 20 ul reaction with T4 DNA ligase (2 ul), rATP (2 ul), and 10x ligase buffer (2 ul).

I had verified the size of the vector and insert that I was going to use in the ligation before I did the ligation, and they were around the expected sizes of 4.5 and 1.5 kb.

Thanks in advance for giving me some advice as to how I should proceed.

GMoorthy

Edited by GMoorthy, 14 April 2009 - 11:26 AM.

[phage434]

NheI and SpeI have compatible ends, and will religate without an insert.  Can you choose enzymes without compatible ends?  I would try to do a lot less handling of your DNA.  PCR the fragments, column purify the DNA to remove the PCR enzymes and dNTPs.  Cut with your enzymes, heat kill (no purification) mix with a molar excess of insert, and ligate.  You'll get no-insert background with this, but it may be at a level that is tolerable.  Gel purification, UV exposure, CIP, and multiple purifications can all cause problems.  Less is more.

[GMoorthy]

phage434, on Apr 14 2009, 05:08 PM, said:

NheI and SpeI have compatible ends, and will religate without an insert.  Can you choose enzymes without compatible ends?  I would try to do a lot less handling of your DNA.  PCR the fragments, column purify the DNA to remove the PCR enzymes and dNTPs.  Cut with your enzymes, heat kill (no purification) mix with a molar excess of insert, and ligate.  You'll get no-insert background with this, but it may be at a level that is tolerable.  Gel purification, UV exposure, CIP, and multiple purifications can all cause problems.  Less is more.

Phage434,

Thanks for replying.  Even if Nhe1 and Spe1 have compatible ends, these ends should not be coming together in the vector if the CIP worked right?  

GM

[perneseblue]

GMoorthy, on Apr 15 2009, 01:20 PM, said:

phage434, on Apr 14 2009, 05:08 PM, said:

NheI and SpeI have compatible ends, and will religate without an insert.  Can you choose enzymes without compatible ends?  I would try to do a lot less handling of your DNA.  PCR the fragments, column purify the DNA to remove the PCR enzymes and dNTPs.  Cut with your enzymes, heat kill (no purification) mix with a molar excess of insert, and ligate.  You'll get no-insert background with this, but it may be at a level that is tolerable.  Gel purification, UV exposure, CIP, and multiple purifications can all cause problems.  Less is more.

Phage434,

Thanks for replying.  Even if Nhe1 and Spe1 have compatible ends, these ends should not be coming together in the vector if the CIP worked right?  

GM

In biology nothing is 100%.

I am concerned that you are seeing ligated DNA but no colonies, even colonies carrying empty vector. How long is your UV exposure time? UV exposure should be measure in seconds not minutes.

You should also check that the NheI site on the PCR fragment has enough bp surrounding it. NheI needs 8bp if I am not mistaken.

[GMoorthy]

perneseblue, on Apr 15 2009, 07:07 PM, said:

GMoorthy, on Apr 15 2009, 01:20 PM, said:

phage434, on Apr 14 2009, 05:08 PM, said:

NheI and SpeI have compatible ends, and will religate without an insert.  Can you choose enzymes without compatible ends?  I would try to do a lot less handling of your DNA.  PCR the fragments, column purify the DNA to remove the PCR enzymes and dNTPs.  Cut with your enzymes, heat kill (no purification) mix with a molar excess of insert, and ligate.  You'll get no-insert background with this, but it may be at a level that is tolerable.  Gel purification, UV exposure, CIP, and multiple purifications can all cause problems.  Less is more.

Phage434,

Thanks for replying.  Even if Nhe1 and Spe1 have compatible ends, these ends should not be coming together in the vector if the CIP worked right?  

GM

In biology nothing is 100%.

I am concerned that you are seeing ligated DNA but no colonies, even colonies carrying empty vector. How long is your UV exposure time? UV exposure should be measure in seconds not minutes.

You should also check that the NheI site on the PCR fragment has enough bp surrounding it. NheI needs 8bp if I am not mistaken.

Yes, you're right, I am not getting any colonies...not even with the empty vector.  I do have 8 bp for Nhe1.  I'm not sure if UV is the issue.  I'm going to try again using low frequency UV or maybe even without UV.  Also, a ligation product > 10 kb or vector concatamers > 10 kb may not be getting into bacteria (I tried using subcloning efficiency competant cells which are probably not great for taking up ligation reactions).  So I'm also trying Top10 cells after making them chemically competant. Will update this forum with my results.

Edited by GMoorthy, 15 April 2009 - 07:10 PM.

[silentears]

GMoorthy, on Apr 15 2009, 03:25 AM, said:

phage434, on Apr 13 2009, 05:20 PM, said:

How are your DNA fragments made?  Cut?  Purified?  How much are you using?  How are you ligating? (temp, enzyme).  We need to know a lot more to debug this.  What controls were run?

Hi, I am having a similar problem.  My vector is around 4.5 kb but after the ligation it runs at >10 kb, suggesting that it forms concatamers.  In the presence of the insert, there is a smear at > 10 kb suggesting some annealing between the vector and the insert is taking place but the vector also seems to be self-ligating. No colonies emerged after transformation.

The vector and insert was PCRed, then gel-extracted and double digested with Nhe1 and Spe1 (15 units of each enzyme for 600 ng DNA) at 37 degrees for 2 hrs. Then both were purified.  Vector was CIPed (0.3 units for 600 ng) in NEB buffer 3 at 37 degrees for 1 hour.  It was purified again before ligation (50 ng vector +/- 250 ng insert) in a 20 ul reaction with T4 DNA ligase (2 ul), rATP (2 ul), and 10x ligase buffer (2 ul).

I had verified the size of the vector and insert that I was going to use in the ligation before I did the ligation, and they were around the expected sizes of 4.5 and 1.5 kb.

Thanks in advance for giving me some advice as to how I should proceed.

GMoorthy

erm can i know wad are the functions of rATP? is rATP = ATP?

[phage434]

Yes, rATP = ATP.  The r is to remind people that although you need dATP in your pcr reaction as a source of nucleotides, you need ATP (which is different!) to drive most enzymatic reactions, such as ligation.

[eldon]

GMoorthy, on Apr 14 2009, 12:25 PM, said:

phage434, on Apr 13 2009, 05:20 PM, said:

How are your DNA fragments made?  Cut?  Purified?  How much are you using?  How are you ligating? (temp, enzyme).  We need to know a lot more to debug this.  What controls were run?

Hi, I am having a similar problem.  My vector is around 4.5 kb but after the ligation it runs at >10 kb, suggesting that it forms concatamers.  In the presence of the insert, there is a smear at > 10 kb suggesting some annealing between the vector and the insert is taking place but the vector also seems to be self-ligating. No colonies emerged after transformation.

The vector and insert was PCRed, then gel-extracted and double digested with Nhe1 and Spe1 (15 units of each enzyme for 600 ng DNA) at 37 degrees for 2 hrs. Then both were purified.  Vector was CIPed (0.3 units for 600 ng) in NEB buffer 3 at 37 degrees for 1 hour.  It was purified again before ligation (50 ng vector +/- 250 ng insert) in a 20 ul reaction with T4 DNA ligase (2 ul), rATP (2 ul), and 10x ligase buffer (2 ul).

I had verified the size of the vector and insert that I was going to use in the ligation before I did the ligation, and they were around the expected sizes of 4.5 and 1.5 kb.

Thanks in advance for giving me some advice as to how I should proceed.

GMoorthy

A 4.5kbp vector plus 1.5 kbp insert should not present any size problems with respect to bacterial transformation. Without electroporation, I routinely transform Top10 cells with 12-15 kbp vectors (including insert).

In my experiences, using CIP or SAP is a gamble as these enzymes can recess the ends of the DNA if conditions are not optimal..(digestions that go to 100% completion don't really exist). You can always get background recircularization in controls where vector is cut with single or multiple enzyme (then dephosphorylated or not) as remaining uncut vector (or singly cut vector in a double digest) is low enough to go undetected by EtBr but efficiently competitive to transform your cells.

One trick I use on occasion is a form of "molecular selection for succesful ligation prior to bacterial transfomation". If your insert is a PCR product that you wish to insert into an NheI/SpeI digested plasmid, rather than using an NheI site on your 5' end of your inserts forward primer, use another RE with compatible sticky ends (e.g., XbaI). After the ligation, take an aliquot equivalent to 2X what you would use for a transformation, dilute the salts if necessary and digest for 1 hr with NheI. This will linearize NheI or SpeI recircularized vector but not vector with insert as ligation of NheI overhang to XbaI overhang destroys the original NheI site within the MCS and thus >90% of colonies will have what you want. This works well for blunt ligation too...especially into SmaI site of MCS.

But...since you are not getting any colonies...I suspect it is your cells. Always include an uncut plasmid control...especially if prepping your own competent cells.

Edited by eldon, 24 May 2009 - 11:43 AM.