No luck with blunt and sticky ligations, need hlep - Prepare for a thorough read (Aug/24/2010 )

Recently I started a short internship in a new lab to familiarize myself with different techniques. I was supposed to generate virus, perform some transductions and analyze cells with FACS and qPCR analyzes. Obviously, the virus first needed to be generated and for this (plasmid) constructs needed to be created.

Collegues in this lab apparently had a hard time cloning sticky-blunt and sticky-sticky, until -after several months- they finally got it to work. Since the vectors I am cloning with have almost no sites left, I need to perform my ligations with blunt-reactions (which I hate). Now, after 1 month having tried several cloning 'improvements' in vain, we seemed to be stuck again in cloning hell.

Short overview of the experiment:
Cutting a 2200bp fragment with BamHI from a 11k vector, blunt
Ligate it in 1 vector that was linearized (ClaI) then blunted and 2 vectors where a small fragment (<500bp) was removed (BamHI&XhoI, NheI&KpnI) then blunted
Ligation is sticky, and I SAP all the vectors prior to ligation.
Transform to competent DH5a E.coli cells with HS method.

The different steps of the experiment explained in more detail + things to improve
Digestion + cutting from gel
I digest for 1-2h, I add SB, run on gel, cut out the fragments and making sure no fragment is exposed to UV for >30s

Purification of fragments from gel
I used the QIAGEN spin colums for purification where I made sure to remove the buffer PE from the collection tube before centrifuging again (so no ethanol was left). I solved in 1x Antarctic Phosphatase buffer (the backbones) and the insert I solve in 1x NEB buffer 2 in the elution step. Lately.

SAP
~20 ul vector is dephosphorylized with 1ul Antarctic phosphatase, 30'@ 37C, subsequent inactivation 15'@ 75C

Analysis and estimation of DNA amount of purified fragments
First 3ul on gel and compared to lambda ladder but lately I have been using the Nanodrop. I seem to be getting concentrations in the range of 15-30ng/ul, A260/280 values range from 1.59-1.90. A260/230 values are 0.08-0.17

Ligation
I ligate insert:vector 4:1 ratio
Vector: 50ng
Vtot = 30ul
Ligation conditions: 16C O/N
I add aliquoted buffer and aliquoted ATP
For overnight ligation I use 1ul T4 Ligase (NEB)

Transformation
I use DH5a chemocompetent cells (Invitrogen)
50ul cells
1-10 ul ligation mix
-30'on ice
-Heat shock 42C 45" (I use 1.5ml eppendorf tubes and a heating block)
-Recovery on ice 2-5 min
-Add 900ul S.O.C. @RT and lately LB medium (we ran out of S.O.C. ) @ RT or 37C, incubate 37C for 1h
-I spread 100ul, I spin down, remove sup and spread remaining on AMP plates @37C

I don't get a single colony!

I started transforming pUC19 plasmid. I transfer 20pg pUC19 to 50ul bacteria. I follow the above protocol and plate 100ul and then get 7 colonies :S. I did a retransformation of a vector I wanted maxiprepped, 1ng in total I get like 500-1000 colonies.

Things I did to fix this (boy, long list)
-After the first failed ligation+transformation, I started using a new vial of T4 ligase for all the experiments. To no avail.
-I used 10% PEG-6000 for blunt ligations overnight, I didn't get any colonies. After this I did not use PEG-6000 anymore
-As the transformation efficiency is quite low, I feared loss of competent cells due to thawing/freezing because of aliquoting the stock. I used a stock-vial but this made no difference!
-I ligated more (200ng vector with 3:1 fragment)and put half on gel. I see that duplos and triplos of my insert but also still see mono insert. The vector band (unligated) is clearly visible but upon closer inspection small bands can sometimes be seen above this (indicating a small amount of insert was ligated in my vector). I transformed 5 microliters of this mix and did not get any colonies!! PEG-6000 seemed to improve the situation on gel but also when I transform it I get no colonies! The weird thing is that, even though a sticky sticky ligation with 2200bp fragment and 7700 bp vector shows vector+insert, there is still a large amount of unligated vector and fragment left. So probably the ligation reaction is not working properly.
-Heat inactivation of T4ligase @ 65C for 5' prior to transformation. No sigar:(
-I walked a vector through this proces and sampled at every step, then ligated these samples without insert. Then I transformed ~50ng of DNA from each of these samples. I got:
97 colonies for untreated vector
71 colonies for cut (unklenoved, unsapped, not run on gel and not purified) ligated vector
2 colonies for cut, klenoved then run on gel and colomn purified vector (but not sapped)
0 colonies for cut, klenoved, gelrun, purified and SAPed vector (expected)
-I redid this reaction where I cut the 11000bp vector with BHI, dont blunt but isolate both the small and the large vector, then I ligate these in 1:1, 1:5, 1:10 with total amount of vector 50ng. I ligated this with 0.5ul t4 ligase @ 25C, subsequently I transform 50ul of chemocompetent E.coli bacteriums with 4ul of this mix, I get zero colonies!!! I did this experiment in duplo, one where I used colomn purified fragments one where I used bead purified fragments (as I have good experience with beads and feared ethanol carryover from column, even though I spun twice and remove the flowthrough) but the result is the same!

One pHD in my group here also had cloning troubles and did a sticky ligation BamHI/EcoRI with 10kb vector and 2kb insert with 50ng vector 6:1. She got 70 colonies. Sounds little to me? A technician did a sticky blunt with 8kb vector and 1.4kb insert and she got 20 colonies:S:S

Still need to check:
-effect of ligation mix on positive control. Maybe something toxic in this mix?
-incubation of bacteria in recovery in 'breathing' tubes (now just in eppendorf tubes with cap closed. this has always worked for me and also here I do get growth)
-We ordered new competent bacteria (maybe we got a wrong batch) so I will check my ligations with this.

Please please please please tell me what I must do to get colonies with my insert!! I can't stand this!

50ng of untreated vector should give you a lawn on your plates...if you're only getting 97, then something is killing your transformation....going from there, it's going to be difficult at best. If you can fix that part, then the rest should be better.

NemomeN007 on Tue Aug 24 14:25:22 2010 said:

Hi Dear,

Dont worry its necessary that some time our experiment should not work in first start as failure teaches us a lot.

Another thing dont waste your time for checking toxicity of ligation etc etc. Just follow few step and i think it will increase your chance to more than 80%. Please follow few suggestions.

There should be cleanlines in your all molecular biology work.

Take care while spreading transformed cells on Agar plate ( Spreader should not be too hot to touch and kill bacteria with heat.

Set up ligation reaction only at 10ul per reaction and transform all mix into bacterial vial. after incubation at 37 degree spin at 3600 rpm for 3 mins and discard all media except 5o ul and resuspend cells in that balance medium with pipette slowly up and down. Plate whole cells on Agar plate.

Please go through the attachment for all detailed protocol.

Please feel free to ask if you have more questions.

Best regards,

Mole .

If you heat inactivate with PEG in your reaction, you will reduce the transformation efficiency.

Hi
From our experience, sometimes gel extraction kit can make troubles with ligations.
I'd recommend trying different kit or different batch.
The rest looks good to me.
Good Luck
Michael

hematopoietry on Tue Aug 24 10:10:21 2010 said:

Recently I started a short internship in a new lab to familiarize myself with different techniques. I was supposed to generate virus, perform some transductions and analyze cells with FACS and qPCR analyzes. Obviously, the virus first needed to be generated and for this (plasmid) constructs needed to be created.

Collegues in this lab apparently had a hard time cloning sticky-blunt and sticky-sticky, until -after several months- they finally got it to work. Since the vectors I am cloning with have almost no sites left, I need to perform my ligations with blunt-reactions (which I hate). Now, after 1 month having tried several cloning 'improvements' in vain, we seemed to be stuck again in cloning hell.

Short overview of the experiment:
Cutting a 2200bp fragment with BamHI from a 11k vector, blunt
Ligate it in 1 vector that was linearized (ClaI) then blunted and 2 vectors where a small fragment (<500bp) was removed (BamHI&XhoI, NheI&KpnI) then blunted
Ligation is sticky, and I SAP all the vectors prior to ligation.
Transform to competent DH5a E.coli cells with HS method.

The different steps of the experiment explained in more detail + things to improve
Digestion + cutting from gel
I digest for 1-2h, I add SB, run on gel, cut out the fragments and making sure no fragment is exposed to UV for >30s

Purification of fragments from gel
I used the QIAGEN spin colums for purification where I made sure to remove the buffer PE from the collection tube before centrifuging again (so no ethanol was left). I solved in 1x Antarctic Phosphatase buffer (the backbones) and the insert I solve in 1x NEB buffer 2 in the elution step. Lately.

SAP
~20 ul vector is dephosphorylized with 1ul Antarctic phosphatase, 30'@ 37C, subsequent inactivation 15'@ 75C

Analysis and estimation of DNA amount of purified fragments
First 3ul on gel and compared to lambda ladder but lately I have been using the Nanodrop. I seem to be getting concentrations in the range of 15-30ng/ul, A260/280 values range from 1.59-1.90. A260/230 values are 0.08-0.17

Ligation
I ligate insert:vector 4:1 ratio
Vector: 50ng
Vtot = 30ul
Ligation conditions: 16C O/N
I add aliquoted buffer and aliquoted ATP
For overnight ligation I use 1ul T4 Ligase (NEB)

Transformation
I use DH5a chemocompetent cells (Invitrogen)
50ul cells
1-10 ul ligation mix
-30'on ice
-Heat shock 42C 45" (I use 1.5ml eppendorf tubes and a heating block)
-Recovery on ice 2-5 min
-Add 900ul S.O.C. @RT and lately LB medium (we ran out of S.O.C. ) @ RT or 37C, incubate 37C for 1h
-I spread 100ul, I spin down, remove sup and spread remaining on AMP plates @37C

I don't get a single colony!

I started transforming pUC19 plasmid. I transfer 20pg pUC19 to 50ul bacteria. I follow the above protocol and plate 100ul and then get 7 colonies :S. I did a retransformation of a vector I wanted maxiprepped, 1ng in total I get like 500-1000 colonies.

Things I did to fix this (boy, long list)
-After the first failed ligation+transformation, I started using a new vial of T4 ligase for all the experiments. To no avail.
-I used 10% PEG-6000 for blunt ligations overnight, I didn't get any colonies. After this I did not use PEG-6000 anymore
-As the transformation efficiency is quite low, I feared loss of competent cells due to thawing/freezing because of aliquoting the stock. I used a stock-vial but this made no difference!
-I ligated more (200ng vector with 3:1 fragment)and put half on gel. I see that duplos and triplos of my insert but also still see mono insert. The vector band (unligated) is clearly visible but upon closer inspection small bands can sometimes be seen above this (indicating a small amount of insert was ligated in my vector). I transformed 5 microliters of this mix and did not get any colonies!! PEG-6000 seemed to improve the situation on gel but also when I transform it I get no colonies! The weird thing is that, even though a sticky sticky ligation with 2200bp fragment and 7700 bp vector shows vector+insert, there is still a large amount of unligated vector and fragment left. So probably the ligation reaction is not working properly.
-Heat inactivation of T4ligase @ 65C for 5' prior to transformation. No sigar:(
-I walked a vector through this proces and sampled at every step, then ligated these samples without insert. Then I transformed ~50ng of DNA from each of these samples. I got:
97 colonies for untreated vector
71 colonies for cut (unklenoved, unsapped, not run on gel and not purified) ligated vector
2 colonies for cut, klenoved then run on gel and colomn purified vector (but not sapped)
0 colonies for cut, klenoved, gelrun, purified and SAPed vector (expected)
-I redid this reaction where I cut the 11000bp vector with BHI, dont blunt but isolate both the small and the large vector, then I ligate these in 1:1, 1:5, 1:10 with total amount of vector 50ng. I ligated this with 0.5ul t4 ligase @ 25C, subsequently I transform 50ul of chemocompetent E.coli bacteriums with 4ul of this mix, I get zero colonies!!! I did this experiment in duplo, one where I used colomn purified fragments one where I used bead purified fragments (as I have good experience with beads and feared ethanol carryover from column, even though I spun twice and remove the flowthrough) but the result is the same!

One pHD in my group here also had cloning troubles and did a sticky ligation BamHI/EcoRI with 10kb vector and 2kb insert with 50ng vector 6:1. She got 70 colonies. Sounds little to me? A technician did a sticky blunt with 8kb vector and 1.4kb insert and she got 20 colonies:S:S

Still need to check:
-effect of ligation mix on positive control. Maybe something toxic in this mix?
-incubation of bacteria in recovery in 'breathing' tubes (now just in eppendorf tubes with cap closed. this has always worked for me and also here I do get growth)
-We ordered new competent bacteria (maybe we got a wrong batch) so I will check my ligations with this.

Please please please please tell me what I must do to get colonies with my insert!! I can't stand this!

did you try Promega ligasfast for 2hr at room temp
mine for some reason did not work for 16°C ON but it qorked fine for 2hr with ligafast

I visualized (sticky) ligations with different V:I ratios (V=8k, I=2k) with fragments purified by beads/column. These fragments perform equally well/ bad. After O/N ligations @ 16C and 25C, a smear was visible above my vector band in the reactions with the highest V:I concentration (5:1, 10:1). I saved 2ul of these mixes for transformation. Also I am preparing ligations with HCC and two step changing j:i ratios, as these are supposed to enhance first intermolecular and then intramolecular reactions.

NemomeN007 on Tue Aug 24 14:25:22 2010 said:

mole on Tue Aug 24 15:23:16 2010 said:

NemomeN007 on Tue Aug 24 15:45:09 2010 said:

ranvi on Tue Aug 24 23:44:44 2010 said:

Hi,

So I've doing a lot of subcloning in the past few years and a few things you said caught my attention.

1). When you AP your vectors, do you do a PCR cleaning up or ethanol precipitation with your DNA before you do your ligation? Usually, when I do ligations, I try to make running the fragments on the gel and then isolating them the last step before the ligation reaction itself so that the stuff in the digestion/AP reactions are removed by the gel extraction process.

2). Also, if you use the Qiaquick Gel Extraction kit, you must let the column incubate at RT for at least 5 minutes after you add the PE (to allow the PE to soaking in and a little bit to run through), this step is critical to removing contaminants that can be detrimental to ligation reactions. In fact, add more PE than what the protocol said, around 850-1000ul, and spin 3 times to get it all out (the last time in a clean eppie) before elution. Eluting with either EB or water is fine for ligation.

3). AP reactions can go for longer than 30 minutes at 37C. In fact, I usually do 40-50 minutes, but 5 minutes at 65C is enough for heat inactivation.

4). I've usually done blunt end ligations using at least 5:1 insert to vector molar ratios, especially if your insert size is large (>1.5-2kb).

5). Although I've read that you should use around 100ng DNA per ligation, I've always had great luck doing 350-400ng DNA in a 20ul ligation reaction, using 1ul of T4 ligase from NEB at 400U/ul. If you use less DNA, you should lower the amount of ligase you use. The reason for this is that ligation will only happen if only one of the two ends to be ligated has the ligase attached. At high concentrations of ligase (compared to DNA), both ends will be occupied by ligase, making the ligation reaction extremely inefficient.

6). If you are doing retroviral/lentiviral vectors with LTRs you should not be using DH5a competent cells as the chances of recombination are high. Stbl3 cells are better. But if you use DH5a, make sure you are using max efficiency, and not subcloning or library efficiency.

7). I usually transform 5ul of a 20ul reaction in 50ul. That works out to be 50-100ng DNA. It's on the upper extremes of what you should be using. The rule I go by is the bigger the plasmid, the less DNA I transform because as you can imagine, the bacteria's ability to uptake DNA decreases with plasmid size and having more DNA will only mean the more competition among plasmids for uptake and less efficiency. It might not be scientific (or even right), but that's how I rationalize it and it hasn't failed me yet.

8). Make sure you are not over-heat-shocking the cells. 30s is enough, 45 secs max. Also, do not shake the cells or disturb it between heat shock and chilling on ice for 2 min. Make sure your competent cells have not been thawed more than once before your transformation. Ideally, you should thaw a 200ul tube, divide in 4, then immediately freeze the other 3 for future use.

9). Do not add more than 3-400ul SOC per 50ul competent cells. You do not need more than 150ul for plating later on and more SOC will not help (and actually, you won't run out of SOC either ). Shake at 37C (212rpm) for at least 1 hour horizontally. Longer shaking (1-2hrs) will help in getting you more colonies, closed tubes are fine. In fact I screw them on as tight as they'll go (so they don't leak out all over the floor of the shaker). Make sure you shake horizontally though (just tape them to the bottom of your shaker).

10). When you are ready to plate, no need for the spinning step, just pipet up and down a few times and plate 150ul per 10cm agar plate. Use sterile, cool spreader (one use disposable ones preferred). Also, try to prewarm your plates in the incubator or at room temp.

If those steps are no good, try using super concentrated ligase with more DNA. NEB sells them at 2,000,000U/ml. Or try using another kit, I hear good things about Roche's fast ligation kits.

As for the effect of ligation mix on transformation. If your ligation mix is just T4 ligase buffer (with ATP), T4 ligase in glycerol, DNA dissolved in EB, Tris-HCl, or water, and molecular grade water, it should not matter at all. PEG is fine too.

Don't give up, these blunt end ligations are super finicky, and it sounds like you are using both large inserts and large vectors which are naturally difficult to do.

Hi SBR, thanks for taking the time to reply,

SBR on Mon Aug 30 00:29:04 2010 said:

SBR on Mon Aug 30 00:29:04 2010 said:

SBR on Mon Aug 30 00:29:04 2010 said:

SBR on Mon Aug 30 00:29:04 2010 said:

SBR on Mon Aug 30 00:29:04 2010 said:

SBR on Mon Aug 30 00:29:04 2010 said:

SBR on Mon Aug 30 00:29:04 2010 said:

SBR on Mon Aug 30 00:29:04 2010 said:

9). Do not add more than 3-400ul SOC per 50ul competent cells. You do not need more than 150ul for plating later on and more SOC will not help (and actually, you won't run out of SOC either ). Shake at 37C (212rpm) for at least 1 hour horizontally. Longer shaking (1-2hrs) will help in getting you more colonies, closed tubes are fine. In fact I screw them on as tight as they'll go (so they don't leak out all over the floor of the shaker). Make sure you shake horizontally though (just tape them to the bottom of your shaker).

SBR on Mon Aug 30 00:29:04 2010 said:

SBR on Mon Aug 30 00:29:04 2010 said: