3-way ligation of 2 annealed dsDNAs and pGEM-T Easy - (Aug/28/2011 )

Hi everyone, I'm having a problem with cloning 2 DNA fragments simultaneously to pGEM-T vector. I hope that someone here can help me to figure out how to solve that. Thank you so much in advance.
My purpose is cloning a DNA fragment of ~140bp to pGEM-T using annealed oligonucleotides. I can order synthesizing long oligonucleotides but the quality is not really good because of error, so I divided it into 2 parts and I ordered 4 oligonucleotides for assembly.
The first part is F1, made by annealing F1-F and F1-R
The second part is F2, made by annealing F2-F and F2-R
The 2 fragments have similar size (about 70bp)

F1-F -------------------------------- F2-F AATG--------------------------------A
F1-R A--------------------------------TTAC F2-R --------------------------------

This is my protocol
1. Anneal F1-F with F1-R and F2-F with F2-R
2. Co-purify the 2 annealed DNA using PCR purification kit
3. Ligate the purified product to pGEM-T

Protocols:
1. For annealing each pair I used the protocol herein:
Mixture recipe:
1. F1-F (200 pmol/ul) 1 ul
2. F1-R (200 pmol/ul) 1 ul
3. EDTA 5mM 10 ul
4. Buffer 3 NEB 2.5 ul
5. Water 35.5 ul

0.5X EDTA (final concentrations: 50 mM NaCl, 25 mM Tris-HCl, 5 mM MgCl₂, 0.5 mM Dithiothreitol)

Thermal program (using PCR machine)

95°C 3’
70°C 10’ Ramp: 0.5°C/s
25°C 20’ Ramp: 0.2°C/s
4°C ∞
2. Purify annealed DNAs by PCR purification kit to remove salts, I mixed the two annealing reactions and purify using one column
I checked the concentration by OD260, it's about 140 ng/ul
3. Ligate to pGEM-T
Mixture recipe:
1. Mixed purified F1/F2 fragments 0.5 ul
2. pGEM-T vector 1 ul
3. 2x T4 ligase quick ligation buffer 5 ul
4. T4 ligase (from the same kit) 1 ul
Keep this reaction at room temperature for 2h, then 16 degree for more 2h before transforming to XL1-Blue cell
I got some white colonies, but after sequencing they were revealed to be unwanted fragments. These clones contained only the second fragment or both but lose the nucleotides at the sticky end (AATG). Seems like this T4 ligase has the fill-in or exonuclease activity? Do you have any suggestion to solve that problem?

Synthesized oligos lack the required 5' phosphate unless it is ordered (extra cost) or added with PNK treatment. You won't be able to ligate anything without doing this.

Thank you for your suggestion, I'm treating the products with PNK now
But how about the exonuclease activity of T4 ligase?
Actually I succeeded many times using this protocol and I also got the vector with insert. The problem is the losing part of the insert.

I would try switching from quick ligase buffer to normal ligase buffer. I've never been a fan of the quick ligase -- it ligates too many things. You might want to reduce the amount of your insert fragments, as well. You want equimolar amounts, and those fragments are very short. Also, low concentrations favor the formation of circular fragments, so I'd aim at around 10 ng of vector and very much lower amounts of both inserts. You could try to visualize the ligation of the inserts.

What I would really do, however, is switch to making the insert with overlapped oligos and using a few cycles of pcr to extend them. A 15-20 bp overlap will prime one against the other, and you'll yield a 140 bp fragment with A tails automatically. You'd save money on the oligos, since they would be single stranded, not double.

phage434 on Mon Aug 29 02:45:22 2011 said:

Sorry, for interrupting here. I get a little confused.
I had done TA cloning, Blunt End cloning and LIC cloning (novagen). I never done PNK treatment in any of my PCR products nor primers. I did not order any special primers either. However, I got my clones done. Did I misunderstood anything here?

Your TA cloning vector had 5' phosphates, so your PCR fragment, despite having no 5' phosphates, was partially ligated (leaving a nick in the other strand). The nick was repaired by enzymes in E. coli after transformation. This won't happen in this case because both strands are missing a 5' phosphate at the junction between the two annealed pieces (unless, as was done, a PNK treatment adds the 5' phosphates).

I did PNK treatment and I got more colonies, even though not much. Finally I got 1 correct among 6 clones (2 of them contained only vector, the other ones had mutations, maybe because of errors in oligonucleotide synthesis.
Thank you so much phage434