Cloning protospacer adapters into
PX330/PX335/PX458/PX459 (Zhang lab, MIT) via BbsI
and bottom oligos of adapter
- Resuspend oligos in water to 1 µg/µl
- Combine the following in a 200 µl PCR tube
- 5 µl “top” oligo
- 5 µl “bottom” oligo
- 10 µl of 10x annealing buffer (10X Annealing
Buffer: 1 M NaCl / 10 mM EDTA pH 8.0 / 100 mM Tris pH
- 80 µl water
- Mix well. Run the mix in a thermal cycler with an
annealing program, such as: Step (1) 94˚ for 3 minutes; Step
(2) cool from 94 ˚ to 25˚ slowly, such as over a 30 minute
period. Alternatively to a thermal cyler: Heat a beaker
of water to boiling. Float the tube in the boiling water bath for
5’. Remove the beaker from the heat and let it cool off naturally
on a benchtop, till it is room temperature.
- Transfer the annealed adapter to a 1.5 ml tube. Add 900
µl water. The final concentration of annealed adapter is now
~ 10 ng/µl.
Ligate to BbsI-cut vector.
- Before you start: You will need to
have the vector DNA previously cut with BbsI and gel-purifed, and
in 10 mM Tris or Lo-TE at a concentration of at least 10
- I use the NEB Quick Ligation kit, and I
reduce the volumes by half to save reagents. It works
great. Combine the following in this order:
- 2.5 µl of 10 ng/µl BbsI-cut vector
- 1 µl of 10 ng/µl annealed adapter
- 1.5 µl of water
- 5 µl of 2x Quick Ligase buffer
- 0.5 µl of Quick Ligase enzyme
- Mix briefly, and incubate at room temp. for
5’. Use immediately for transformation.
- You will need a 42˚ water bath and LB+AMP
plates. You will also need to get a tube of competent
DH5α cells from the 9th floor core in Light
Hall. Thaw the cells on ice.
- Transfer 100 µl cells to a 1.5 ml tube.
- Add 5 µl of the ligation reaction. Mix
well (do not vortex).
- Incubate 45’ on ice.
- Heat-shock the cells at 42˚ for 2’.
- Transfer cells back to ice.
- Add 900 µl of LB media to the cells.
Transfer the cells to a 15 ml tube.
- Recover the cells by incubating in a 37˚
shaker incubator for 30’ at 250 rpm.
- Plate out 100 µl of the cells on
an LB+AMP plate. Incubate at 37˚ overnight. You should get at
least a few dozen colonies (e.g. 10-100 is typical). Most of them
will contain the correctly cloned product. When I have
done negative controls with no adapter, I get zero or just a couple
Screen colonies for correct adapter
- Inoculate 2 colonies separately into 15 ml tubes with 2
mls of LB+100 µg/ml AMP. Shake overnight at 37˚.
- Perform Qiagen minipreps on 1.5 ml of each plasmid
culture. Save the remaining ~0.5 ml for making a glycerol
stock to store at -80˚. (see below). The culture sample can
be stored at 4˚ for a few days before you make the glycerol
- Spec the DNA. Usually the concentration is about
40-100 ng/ul. Prepare a sample for direct Sanger
sequencing using this primer:
This sequencing will read-through the region of the BbsI site
and verify that the adapter has inserted properly.
Make glycerol stocks of the correct
- To the remaining ~0.5 ml of the miniprep culture in step
IV above, add 0.5 ml of sterile 30% glycerol. (Glycerol
solutions should be sterilized by filtration, never
- Mix and store the glycerol stock at -80˚.
Doug Mortlock 7/23/14