Naegleria 2022
Naegleria 2022 kdorfman Fri, 12/17/2021 - 21:18schedule ((Digest+CIP, column purify, ligation, transformation). Flowcharts+review+revision**
date | # | topic |
---|---|---|
1/26 | 1 | Intro |
1/31 | 2 | Microscope bootcamp DAPI slides, fluorescence microscopy power point |
2/2 | 3 | 2/2 | 3 | Cloning 1 ((Digest+CIP, column purify, ligation, transformation). Flowcharts+review+revision |
2/7 | 4 | Naegleria 101 |
2/9 | 5 | Cloning 2 |
2/14 | 6 | Experiment 1 |
2/16 | 7 | Experiment 2 |
2/22 | 8 | Experiment 3 |
2/23 | 9 | Wrap up |
Competent HT115
Competent HT115 kdorfman Mon, 12/20/2021 - 15:59Inoculate overnight culture in LB + antibiotic (TET for HT115(DE3) strain) (2-5ml). Shake overnight at 37C.
Inoculate 25 ml LB + antibiotic with overnight culture, 1:100 dilution. Grow cells to OD595= 0.4. Can grow cells in 50 ml sterile centrifuge tube.
Spin cells 10 min 3000 rpm at 4C.
Resuspend pellet in 0.5X original volume cold, sterile 50 mM CaCl2 (12.5ml). Resuspend by GENTLY pipetting up and down a few times with a wide bore pipet--no vortexing.
Incubate on ice 30 min.
Spin as before at 4C.
Resuspend pellet as before in 0.1X original volume CaCl2 (2.5ml). Keep cells cold (4C).
Use 50-200 ul for transformation. Cells can be used as is for up to three days (stored at 4C).
The cells can be frozen by: adding glycerol to final concentration of 10%, rapid freezing on dry ice/EtOH, and storing at –80.
Naegleria 3
Naegleria 3 kdorfman Sat, 12/18/2021 - 12:00Cloning Bootcamp
- 37C heatblock
- 42C heatblock
- Age1
- 10X buffer NEB1.1
- rSAP
- Sterile water for cloning
- Ligase + 2X rapid lligase buffer
- 1 sample: PCR cleanup kit (Qiaquick) (one per group)
- 1 tube (5 rxns) Competent cells (DH5 alpha, just for cloning)
- 5x LB+Amp plates
- Sterile beads for spreading
- 30 ng/ul backbone plasmid for cutting (estimated 15n/ul after elution w/ 30ul)
- Gel purified inserts (gamma tub, centrin, sas-6 at 15ng/ul)
- LB (plain) for transformation recovery
Naegleria 4
Naegleria 4 kdorfman Sat, 12/18/2021 - 12:37Naegleria 101 (2/7/22)
Activities:
- Differentiate cells,
- watch amoebae (phase microscopy),
- look at flagellates.
- Pick colonies for cloning.
Materials:
- 4 LB+ Amp +
- Tubes for growing cultures from previous lab’s cloning
- Toothpicks for above
Naegleria 5
Naegleria 5 kdorfman Tue, 02/08/2022 - 21:088 QIAprep Spin miniprep kits for 4 cultures each
Notes before starting
- Optional: Add LyseBlue reagent to Buffer P1 at a ratio of 1 to 1000.
- Add the provided RNase A solution to Buffer P1, mix and store at 2–8°C.
- Add ethanol (96–100%) to Buffer PE before use (see bottle label for volume).
- All centrifugation steps are carried out at 13,000 rpm (~17,900 x g) in a conventional table-top microcentrifuge.
Each kit contains:
- 4 spin columns
- 6 buffers, as below
Buffer | uL per rxn | mL per 4.5 rxns |
---|---|---|
P1 | 250 | 1.125 |
P2 | 250 | 1.125 |
N3 | 350 | 1.575 |
PB | 500 | 2.25 |
PE | 750 | 3.375 |
EB | 50 | 0.225 |
Other materials
- Age1 + cutsmart
- 8 2mL tubes of LB broth
- competent HT115 cells from -80
- two 1% gels for wide gel rig, 2 small-well combs each
- 1X TAE running buffer
- 1KB DNA standard
Equipment
- Microcentrifuges capable of 13000rpm
- Hot blocks at
- 37
- 42
- two wide gel rigs
- two power supplies
- two blue light boxes with photography box
Naegleria 6
Naegleria 6 kdorfman Thu, 02/10/2022 - 14:06M Feb 14
32 (?) tubes of LB-amp