ligation

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scchaudhary
scchaudhary's picture
ligation

Hello!
i have problem for ligation for 2 months. 'plz help and suggest me.
now , i wanted to just chek whether the ligATION is working or not.
i wanted to ligate avian RSK1 into PKH3 vector .for this i cut both with BamH1 and Hpa1 enzyme .
i cut two bands in gel electrophoresis.  i also ligated both cuts ( BANDS) of PKH3 as a positive control and AND ALSO pkh3 as vector and RSKN1 as insert.
i did transformation.
Unfortunately i did not get any colony.
i suffered this for 2 month .
i couldnot find which step is wrong and where is the proplem? i got frusted . Please suggest me

The FFM
The FFM's picture
my gosh..where to start!

my gosh..where to start!

Your restrictions enzymes seem like thery're cutting ok, but you will need to double check.   Your DNA ligase might be bad.  Your transformation efficiency may be very poor. The DNA purifications from the agarose gels may be bad. 

Here are some basic steps to start, given the information tha you have provided.

1) Confirm that performing single cuts with either BamHI or HpaI is giving you the correct size fragments on an agarose gel for both vector backbone and insert. Then perform the double digest and isolate the bands.  perhaps one of your enzymes is not cutting properly and you dont have the correct sites available for the ligation reaction to work.

2) Check the yields of the bands after you have purified them from the agarose gel (and the purity A260/280).  Then Typically you will need to include a 3:1 molar ratio of insert to vector in the ligation reaction for success.

3) Make sure you perform a no insert negative control ligation on a dephorphorylated vector

4) Perfrom a positive control ligation on a single cut dephosphorylated vector

5) Perfrom a control transformation with an uncut vector to check efficiency

6) Make sure you are using the correct Antibiotic selection!

7) What is the source of the Avian RSK1?  are you cutting it form another plasmid or are you PCRing the template and then digesting it?  If you do not leave enough flanking sequence on a PCR fragment, sometimes the enzymes have a hard time digesting the ends properly.

scchaudhary
scchaudhary's picture
hello!

hello!
Thanks alot for good suggestion.
previously i did pcr and isolate 658 bp inser from pmb1 puro vector .this 658 bp was ligated to TA vector followed by transformation, mini prep , dna isolation and then cut by MFE1+Hpa1 and isolated 658 bp insert on gel lectrophoresis the bands were found on right.In second step I cut puro vector withMfe+hpa1 and delet 1114bp and isolated 8479bp ,In this 8479 bp (puro cut) , I inserted( ligated)  658 bp already obtained from TA vector  followed by transformation where i didnot get many colonies just (7-8 colonies ) after mini prep , dna isolation and cutting by same enzyme , i  got no result . i did this 3 times but the result was same so i try to check whether there is ligation problem or some any other step , so i did ligation  taking pkh3 vector of 4.2 kbp and insert of 600bp of same pkh3 as +ve contol AND 4.2kb pkh3 as vector and 2.8 insert of avian rsk1(http://www.addgene.org/pgvec1?f=c&cmd=findpl&identifier=8998) but i didnot succed as mentioned already. so i need more help and suggestion from any of you .
I would like to thanks again for replying my first question

Jason King
Jason King's picture
I would always recommend

I would always recommend running a quarter of your ligation reaction on an agarose gel after the overnight incubation. If the ligation has worked you will see multiple bands (not just 2) and you should be able to figure out which band corresponds to your correct ligation.

If the ligation has worked then your next focus should be the competent cells. I guess these are not performing as they should. Using a control (1ng of pUC or pBS) you should be able to get at least 1000 colonies. If the cells are good then this number should be over 10,000 colonies per ng.

scchaudhary
scchaudhary's picture
hi!

hi!
First of all I would like to thanks  all of you for giving  me right suggestion for the protocol and methods. now i am working accordingly strictly following your methods.

Thanking  you again

scc