Amplification of concatenated linear ligated fragments - (Dec/11/2010 )

Hi everybody,
I have a linear ligation product composed of 2 pieces (about 3kb in size). When I ran the ligation reaction on the gel, there were some unspecific bands as well as a faint band of around 3kb which seems to be the ligated band of interest. So I tried to amplify the 3kb band using Forward primer of the 1st fragment and Reverse primer of the 2nd one. Unfortunately I get only smear after amplification. Even changing annealing temperature (from 60-70) didn't affect positively.
I use high fidelity Phusion polymerase which recommend higher Ta (Tm+3), however it doesn't work. Also I tried different Mg concentration, but without promising results.

Tm(F)= 68.8
Tm(R)= 67.3

the questions are;
1. How can I ligate two or more linear fragments much more effectively and with higher yield for band of interest?
2. How can I amplify the ligated band of interest which is faint?

Should it seem possible to you kindly help me to find the solution.

many thanks

You'll either need to isolate the faint 3 kb band from the ligation mixture by gel purification and use that as a PCR template, or clone the fragments into a vector and do colony PCR to find clones that contain the correct fragment.

skad on Sat Dec 11 11:38:28 2010 said:

1. What you need is a termination fragment to terminate the ligation reaction. ie you need a vector fragment to ligate to the ends of the two inserts and form a circular plasmids (that you can then later amplify). Without a termination fragment (plasmid vector), the ligation reaction will continue and continue. It should be noted that a ligation between insert A and insert B can produce three initial product, insertA-insertA, insertB-insertB and insertA-insertB. All three initial product will amplify with PCR using primers for A and B Thus PCR is of no use here.

2. Ligate the two inserts into a plasmid (multiway ligation) and transform said plasmid into cells. screen for a colony containing the two inserts. Grow said colony and extract the plasmid. Lot of product.

Hi Veteran, tnx for your reply,
I already did ur suggestion, but the result was smear and when I tried different strategies to optimize PCR and remove smears, there were not any band and just some very short unspecific bands.

Tnx "Unlimited ligation works!"
sorry I didn't get what you recommend about termination fragment.
but I think among the initial products when forward primer for A fragment and reverse primer for B fragment is used in PCR, only insertA-insertB will be amplified. because both of the insertA-insertA and insertB-insertB doesn't have any priming site for second primer. Am I right?