C. elegansC. elegans kdorfman Mon, 01/09/2012 - 17:15
Protocol for making competent cells:
Bacterial strainsBacterial strains kdorfman Wed, 01/18/2012 - 21:58
|Bacterial Strain||Strain Description||Details|
|HT115(DE3)||HT115(DE3) E. coli||RNAi feeding strain|
|HT115(DE3)/pL4400||Empty RNA1 feeding vector L4440 in feeding strain||Ampicillin selection No phenotype|
|HT115(DE3)/pL4400(bli-1)||blister-1 RNAi feeding strain||induces blister by RNAi|
|HT115(DE3)/pL4400(dpy-10)||dpy-10 RNAi feeding strain||induces dumpy by RNAi|
|HT115(DE3)/pL4400(rol-5)||rol-5 RNAi feeding strain||induces roller phenoytpe by RNAi|
|HT115(DE3)/pL4400(T04A11.6)||him-6 RNAi feeding strain||induces high incidence of males by RNAi|
|HT115(DE3)/pL4400(unc-22)||unc-22 RNAi feeding strain||induces twitcher by RNAi|
|HT115(DE3)/pL4400(unc-23)||unc-23 RNAi feeding strain||induces uncoordination by RNAi|
|OP50||Uracil auxotroph E. coli B.||feeding behavior for C. elegans|
Competent cellsCompetent cells kdorfman Thu, 01/19/2012 - 06:14
Need competent cells for students in week 3 - make them in week 1 or 2.
0.5 M PIPES disodium salt(pH 6.7) (Alfa Aesar 3p B21835-22) or Fisher AC21509-1000
- 17.3 g PIPES (disodium salt) in 80 mL H2O
- OR 15.1 g PIPES in 80 mL H2O
- initial pH = ~5.5
- KOH pellets till it goes clear (checking pH!!)
- final pH to 6.7 with 5M KOH
- water to 100 mL
- filter sterilize
- freeze in 10 mL aliquots (enough for 500 mL transformation buffer)
|Reagent||cf (mM)||per L||.|
|PIPES (pH 6.7, 0.5M)||10||20||mL|
filter sterilize. refrigerate
- 950 ml of deionized H2O
- 20 g Tryptone
- 5 g Yeast Extract
- 0.5 g NaCl
- stir to dissolve
- 2.5 mL 1M KCl (10 mL 250 mM KCl)
- pH to 7 with NaOH
- volume to 1 L
- autoclave 20 min
- 10 mL sterile 1M MgCl (5 mL sterile MgCl2 2M)
- ? 10 mL sterile 1M MgSO4?
15 g agar/L SOB
when cooled, add
20 mL 1 M MgSO4
12.5 µg/mL tetracycline (=0.83 mL 15 mg/mL tet per L)
50 µg/mL ampicillin as needed (= 1mL 50 mg/mL amp stock per L)
LB ampLB amp kdorfman Wed, 02/08/2012 - 15:40
LB ampicilin plates for transformation
48 plates x 25 mL/plate = 1200 mL
Make 1500 mL:
25 g LB + 15 g agar /liter and 50 µg/mL ampicillin
37.5 g LB in 1500 mL water in a 2L flask
stir to dissolve, leave stir bar in.
add 22.5 g agar
Autoclave 50 min. Include PourBoy tubing if necessary, and 2 flasks of water.
Cool till handle-able
Add 1500 µL 50 mg/mL amp
Dispense 25 mL/plate
NGM platesNGM plates kdorfman Mon, 01/09/2012 - 17:26
Per Liter of medium (~75 plates):
- 975 mL Water
- 3 g NaCl
- 2.5 g Peptone (Fisher BP1420-500 $78.80)
- 17 g Bactoagar
This is what I did by mistake: per 1.5 L (112 plates):
- 1462 mL Water
- 18 g NaCl
- 15 g Peptone (Fisher BP1420-500 $78.80)
- 25.5 g Bactoagar (haven't added it yet)
So I labeled it NGM 4X.
I made 4 liters by diluting 975 mL 4xNGM with 975 x 3 mL water, then added 68 g bactoagar.
Stir flask to distribute ingredients.
Autoclave with stir bar inside
Cool to 55C in a 55C water bath
Add per L (see recipes in stock solutions):
- 1 mL cholesterol (5 mg/mL in 95% EtOH)
- 1 mL CaCl2 (1 M, STERILE)
- 1 mL MgSO4 (1 M, STERILE)
- 25 mL K-phosphate buffer (1M, pH 6.0, STERILE1)
Swirl flask to mix
Dispense 10 mL into each 60mm dish.
Stack 10 high
Let stand for ~48 hours for condensation to evaporate
Pack in sterilized plastic boxes.
3.3 mL K2HPO4 + 21.7 mL KH2PO4 ↩︎
OP brothOP broth kdorfman Tue, 01/10/2012 - 16:15
B Broth (for OP50 E. coli to feed to worms)
- 10 g bactotryptone
- 5 g NaCl
- 1 L dH2O
400 µL overnight culture to seed plates.
Store for months in refrigerator.
200 mL cells ON for 500 plates
(can save the extra ON culture in the refrigerator for 3 days.)
3.1. Preparation of bacterial food source
Although C. elegans can be maintained axenically (Avery, 1993), it is difficult, and the animals grow very slowly. C. elegans is usually grown monoxenically in the laboratory using E. coli strain OP50 as a food source (Brenner, 1974). E. coli OP50 is a uracil auxotroph whose growth is limited on NGM plates. A limited bacterial lawn is desirable because it allows for easier observation and better mating of the worms. A starter culture of E. coli OP50 can be obtained from the CGC or can be recovered from worm plates. Use the starter culture to isolate single colonies on a streak plate of a rich medium such as LB agar [10 g Bacto-tryptone, 5 g Bacto-yeast, 5 g NaCL, 15 g agar, H2 O to 1 litre, pH 7.5] (Byerly et al., 1976). Using a single colony from the streak plate, aseptically inoculate a rich broth, such as L Broth [10 g Bacto-tryptone, 5 g Bacto-yeast, 5 g NaCl, H2 O to 1 litre, pH to 7.0 using 1 M NaOH. Put 100 ml into 250 ml screw-cap bottles and autoclave. The bottles of media can be stored at room temperature for several months (Byerly et al., 1976)]. Allow inoculated cultures to grow overnight at 37°C. The E. coli OP50 solution is then ready for use in seeding NGM plates. The E. coli OP50 streak plate and liquid culture should be stored at 4°C and will remain usable for several months.