| Bacteria-mediated RNAi | | 点击： 作者：51protocol收集 来源： 时间： 2007-03-28 本站论坛 |
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Transformation of CaCl2 competent HT115(DE3):
1) Add 50-200 ul competent cells to cold, sterile, polypropylene tube on ice.
2) Add 1 uL plasmid (mini-prep quality is OK) 1-100ng.
3) Incubate on ice/water bath for 30 min.
4) Immerse tube in 37C water bath for 1 min.
5) Incubate tube on ice/water bath for 2 min.
6) Add 1ml sterile SOC media. Incubate 37C with shaking for 1 hour.
7) Plate 10uL, 100uL, 250uL, and remaining culture onto 4 LB tetracycline other antibiotic? (plasmid resistance) plates. Incubate 37C overnight. (These cells grow slowly, allow 36 hours for colony formation.)
Freezing bacterial stocks:
1) Inoculate fresh single colony of bacteria into 2.5 ml LB antibiotic(s). Grow to early stationary phase.
2) Pipette 0.25 ml 80% glycerol (sterile) and 0.75 ml culture into a sterile screw-cap freezer tube. Mix.
3) Quick freeze on dry ice/ethanol and store at -80C.
Induction of dsRNA in HT115(DE3) cells T7 promoter containing plasmid:
1) Inoculate overnight culture of HT115(DE3) plasmid in LB antibiotics. Incubate 37C with shaking overnight. (75-100ug/ml ampicillin for amp-resistant plasmids and 12.5 ug/ml tetracycline.)
2) Dilute culture 1:100 in 2xYT antibiotics and grow to OD595=0.4. (A 25ml culture is usually enough for a small experiment ~ 20 small plates.)
3) Induce by adding sterile IPTG to 0.4 mM. Incubate 37C with shaking ~ 4 hours (see note).
4) Spike culture with additional antibiotics (another 100ug/ml AMP and 12.5ug/ml TET) and IPTG (to final total concentration of 0.8mM). Seed plates using culture as is or concentrate cells by centrifugation as per general protocol.
note: the same parameters which give variable results in protein expression using the T7 promoter system are also variable in this system. Variables to consider: induction temperature (37C vs 30C), induction time (2hr, 4hr, overnight--although overnight induction never works for me), concentration of IPTG, induction volume, media (LB vs 2xYT or other medias), additives to induction media (uracil, lactose,etc), etc. Alterations from this "standard" protocol have not significantly or reproducably improved the RNAi results for the genes we have tested. In addition, ÒfreshÓ cells tend to work best. Bacteria that have been stored on plates at 4C for a long period of time often lose effectiveness: try streaking a new plate from frozen cells plasmid or try retransforming HT115(DE3) with the plasmid of interest.
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