11 replies to this topic

[sagar]

hi there

im in a big problem .i have been doing this ligation experiment for 1 month and still have no result in hand.i m trying to clone a 1.4 kb gene into a 2.8 kb vector.both of them are cut with ecori and hindiii of fermentas.The t4 dna ligase is also of fermentas.i have been trying in all molar ratios ie1:3,1:5 ratio of vector:insert in 4C,16C and room temperature but nothing helps.im really running out of time.

Can anyone plz give me any suggestions for this process?what should i do such that i get this ligation coz i think there is no problem with competent cell
Thanks in advance.

[Dr Teeth]

More information would be helpful.  Where is the problem?  Are you getting religated vector? no colonies? colonies without vector? incomplete digestion? etc.

[sagar]

Dr Teeth, on Mar 10 2009, 01:05 AM, said:

More information would be helpful.  Where is the problem?  Are you getting religated vector? no colonies? colonies without vector? incomplete digestion? etc.

hi

yes im having problem in getting colonies ,not even a single colony do i get.in order to check the efficiency of the competent cells i transformed it with a known plasmid and the efficiency was okay.thus i think that there is no problem with transformation.even if there is self ligation of vector still ill be getting single colonies after transformation im nt getting that also.its simply that ligation is not working.
regarding digestion of vector and insert ,its complete as observed in gel.

i wonder how should i do this ligation.
thanks in advance.

[rkay447]

Few questions: what conditions are your digestion?  Are you using the EcoRI buffer?  I use NEB enzymes so I'm not sure what the suggested buffer for fermentas is.  My main question is with the insert.  Is this a pcr product?  If so, does your primer have at least three residues before the Hind III digestion site?  Check the "cleavage close to the end of dna fragments" in the NEB catalog.  For Hind III it says that without three extra residues you get absolutely no digestion of the insert.  In a two hour digestion it says you only get 10 percent.  20 hour digestion results in 75%.  Perhaps your insert is not digesting well (or at all) for Hind III.  

How much vector DNA are you using in the ligation?  I've found that more is not always better.  I only use 25ng of vector for a ligation.  With the sizes of DNA you are working with, that means a 3:1 ratio with 25ng vector should have around 37.5ng insert.  Is this the same amount you are trying?  I've just seen many people miscalculate a ratio.  

The only other thing I can think of is: how much of the ligation are you trying to transform?  Again, more is not always better.  I've seen a ligation transformation of 6ul result in no colonies while a 2ul transformation of the same reaction gave multiple, positive colonies.

[yja97]

I guess there is a problem in your transformation rather than the ligation. I suggest you to check..

1. Antibiotics : Make a new stock. Maybe concentration matters if you're using a low copy number plasmid. You may transform your vector as an uncut form.

2. Ligation DNA: DNA could be degraded before ligation during enzyme cutting. Check your DNA in an agarose gel before ligation.

3.And this is rare case but important.. is the Ori of your plasmid correct?

[almost a doctor]

2 questions about your ligation.

1. Are you dephosphorilating your vector?

2. do you have a positive control, i.e. are you sure your T4 ligase works?  

If RE is working, and transformation is working, I'll check your ligation reagents before repeating and repeating.

hope this helps

[sagar]

rkay447, on Mar 10 2009, 01:35 AM, said:

Few questions: what conditions are your digestion?  Are you using the EcoRI buffer?  I use NEB enzymes so I'm not sure what the suggested buffer for fermentas is.  My main question is with the insert.  Is this a pcr product?  If so, does your primer have at least three residues before the Hind III digestion site?  Check the "cleavage close to the end of dna fragments" in the NEB catalog.  For Hind III it says that without three extra residues you get absolutely no digestion of the insert.  In a two hour digestion it says you only get 10 percent.  20 hour digestion results in 75%.  Perhaps your insert is not digesting well (or at all) for Hind III.  

How much vector DNA are you using in the ligation?  I've found that more is not always better.  I only use 25ng of vector for a ligation.  With the sizes of DNA you are working with, that means a 3:1 ratio with 25ng vector should have around 37.5ng insert.  Is this the same amount you are trying?  I've just seen many people miscalculate a ratio.  

The only other thing I can think of is: how much of the ligation are you trying to transform?  Again, more is not always better.  I've seen a ligation transformation of 6ul result in no colonies while a 2ul transformation of the same reaction gave multiple, positive colonies.

hi there
actually i have tried the digestionn in a common buffer for both the enzymes as per instructions from fermentas.
Intially i check that digestion is working or not by first performing single digestion with hindiii and ecori for 12hrs and run in gel .usually i'll load uncut plasmid to confirm whether its getting digested or not and all the time i get bands for the required plasmid at around 2.8kb .so may be there is no problem with digestion .
After elution of the final digetsion i get vector and insert concentration around 50ng/ul.i tried with 3:1 and 5:1 ratio for insert:vector but it just doesnt work.i transform an uncut plasmid as a positive control and i get colonies but using the same plasmid in cut form i dont get any colony.
What should i do?

[phage434]

Do you gel extract these insert?  If so, beware of UV damage to your DNA.

I would test the competency of my cells.  You should be getting a lawn of transformants from uncut plasmid.  Dilute uncut plasmid to 10 pg/ul and transform 1 ul.  You should get hundreds of colonies.

I would try religating your singly-cut vector.  This will test your ligase and DNA purification issues.

[swanny]

Also religate insert to make sure both sites are good. You should see dimer, trimer etc. Religate DNA ladder in a similar way (~20 minutes at RT) in the same way to make sure the ligase is good. How old is the buffer; has it had lots of freeze-thaws? the ATP can go off, so try fresh ligase and buffer (you can also aliquot the buffer to reduce the numebr of freeze-thaw cycles).

[sagar]

phage434, on Mar 11 2009, 01:56 AM, said:

Do you gel extract these insert?  If so, beware of UV damage to your DNA.

I would test the competency of my cells.  You should be getting a lawn of transformants from uncut plasmid.  Dilute uncut plasmid to 10 pg/ul and transform 1 ul.  You should get hundreds of colonies.

I would try religating your singly-cut vector.  This will test your ligase and DNA purification issues.

thanks
i would try to religate my vector .its having ecori site at one end and hindiii at the other end should i use only this vector or take the same vector cut with only one enzyme either hindiii or ecori.this is good to check efficiency of ligase action.

[namitha]

hi this is namitha.. im doing porject on cloning, expression and purification of GST from proteus mirabilis into E.coli. im doing from past 3 months..still im stuck with cloning itself. i dont know y am not abel to produce clones. im using pET32a vector for cloning GSt gene . the gene is abt 26k da. and pet 32 a iv been digesting it with ECO R1 and HIND III . im getting all restriction correctly and even after ligation im geting the colonies only in expected ones.the control doesnt have colonies and also the CC amp+ . then after isolation of plasmids we do pcr screening with GSt primers to confirm the presence of clones but in that step alone im not getting amplification my positive control gets amplified int hat step..but i did this trice till now. still the same result. i transformed it in BL21 E.coli cells....


if anybody knows anything regarding this plzzzzzzzzzzzzzzzzzzzzzzzzzzzzz let me know...i just have 2 more weeks to complete  expression also,.. im already out of time..please some one help me out..i willl be very gratefull...

[sagar]

namitha, on Mar 12 2009, 02:00 PM, said:

hi this is namitha.. im doing porject on cloning, expression and purification of GST from proteus mirabilis into E.coli. im doing from past 3 months..still im stuck with cloning itself. i dont know y am not abel to produce clones. im using pET32a vector for cloning GSt gene . the gene is abt 26k da. and pet 32 a iv been digesting it with ECO R1 and HIND III . im getting all restriction correctly and even after ligation im geting the colonies only in expected ones.the control doesnt have colonies and also the CC amp+ . then after isolation of plasmids we do pcr screening with GSt primers to confirm the presence of clones but in that step alone im not getting amplification my positive control gets amplified int hat step..but i did this trice till now. still the same result. i transformed it in BL21 E.coli cells....


if anybody knows anything regarding this plzzzzzzzzzzzzzzzzzzzzzzzzzzzzz let me know...i just have 2 more weeks to complete  expression also,.. im already out of time..please some one help me out..i willl be very gratefull...

hi there
looks like u r also having the same problem.like u said your colonies are oming after ligation but in pcr its not giving positive result.either these may be fake colonies it might happen wen u keep amp plate for more than 12 hrs in incubator.why dont u do digestion ?in that way u can confirm whether the colonies u r getting are positive or not .
hope this helps.