Naegleria 2022

Naegleria 2022 kdorfman Fri, 12/17/2021 - 21:18

planning page

schedule ((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 - 15:59
to make: aliquots
at this volume: uL
Innoculate mL LB tet
with mL overnight culture
Grow to OD595=0.4
Spin 3000rpm for 10 min at 4C
Resuspend in: mL cold 50mM CaCl2 GENTLY
Spin as before, 3000rpm for 10 min at 4C
Resuspend in: mL cold 50mM CaCl2 GENTLY
Use immediately or freeze by adding: mL sterile glycerol

  • Inoculate 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 - 12:00

Cloning 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 - 12:37

Naegleria 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 - 21:08

8 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

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 - 14:06

M Feb 14

32 (?) tubes of LB-amp