Annealing and cloning of four single stranded oligos - (Feb/05/2007 )
I have a problem with a tricky cloning procedure.
My plan is to anneal four ss-oligos, and then clone them into a vector double digested with ClaI and EcoRV.
The oligos are all quite long (~60 bp), and when annealed will form a ~130 bp block with the blunt-ended EcoRV site and the sticky end of ClaI.
The strategy i have tried so far is this, i mix the oligos (100nM each) in 20 uL with 1x One-Phor-All PLUS Buffer(Amersham), i then heat the mixture to 94 C for 10 min followed by slow cooling to room temperture (by unplugging the heat-block). I then mix 10,5 uL of this mixture with 2.5uL of double-digested vector and add T4 ligase +T4 ligase buffer for an 15 uL ligation reaction.
I typically ligate for 24 h at 16 C and then transform the entire ligation mixture into competent E.coli cells. But upon screening i only get colonies without insert. I always do a control transformation without insert to keep track on how complety digested my vector is. In this case the amount of colonies seems higher on the plate with insert.....
Do anyone have a good protocol for this kind of procedure or any tips for any of the steps i have described??
Any help would be greatly appreciated!
1. I typed in the sequence of the first oligo (Bio-1) into the Oligo Analyzer program at www.IDTDNA.com, and hairpin analysis indicates that it can potentially form a large stem-loop by hybridinzing to itself. This would prevent it from hybridizing to the second oligo. I assume that the other three probably have the same risk.
2. Rather than trying to hybridize all 4 oligos simultaneously, I would try 1 and 2 in one reaction and 3 and 4 in a second reaction, combining them afterwards in the ligation reaction. I would also recommennd quick cooling the 94C reaction on ice.
3. Otherwise, reduce Bio-2 and Bio-3 to 18 mers and anneal them to Bio-1 and Bio-4, respectively. Change Bio-4 by eliminating the terminal 5' CG to go for EcoRV on both ends, or add a GC-rich three base clamp to the end so that you can obtain the Cla I overhang by restriction. Primer extend to fill in the template, restriction cut Bio-3/4 if necessary, and then combine 1/2 with 3/4.