Sam’s molecular biology protocols
pQuadra RNAi primer design
Notes
pQuadra is a simple and fast plasmid system for cloning stem loop RNAi constructs.
It uses an interrupted type II RE such that the overhangs can be defined by the user.
This facilitates a 4-way ligation using a single amplicon combined with a stuffer
fragment and vector.
Do all bacterial transformations using recombination deficient bacteria (not XL1 blue
cells – they occasionally recombine out the stem loop). Verify the final midiprep (as
well as the Miniprep) does indeed contain the stem loop.
Reference:
Inoue, M. et al. The 14-3-3 proteins of Trypanosoma brucei function in motility,
cytokinesis, and cell cycle. J Biol Chem 280, 14085–14096 (2005).
Protocol:
1. Design primers to amplify the fragment of DNA that will be used for RNAi
a. RNAit is an automated RNAi primer design program that will facilitate
this: http://trypanofan.path.cam.ac.uk/software/RNAit.html
b. Ensure that as much of the gene sequence as possible is used for RNAi
i. Ideally about 1kb
c. Keep the other defaults unchanged
2. Verify that the resulting amplicon does not contain a BstXI restriction enzyme
site
a. Use A Plasmid Editor (APE) or other cloning program to help with
this
3. Add the following sequences to the 5’ end of each primer:
FOR: ataccaatgtgatgg
REV: ataccatagagttgg
4. Digest the following with BstXI
a. pQuadra 1 (a plasmid with the “loop” of the stem-loop)
i. releases 501nt
b. pQuadra 3 (the vector backbone)
i. backbone is 5558nt
c. the RNAi amplicon
i. about 1kb
5. Gel purify the digested fragments and ligate together
6. Ligate the fragments so that the ratio is:
a. amplicon (2) : pQuadra1 (1): pQuadra3 (0.2)
7. Screen for +ves by colony PCR using the RNAi primers
8. Set up an overnight culture from 3 positives
9. Re-screen the positives by restriction digest
a. HindIII releases the fragments and the loop together
b. BstXI releases the fragments and the loop as separate fragments