Animal Cap Experiment

Cutting Caps

  1. Culture in ficol until stage 8
  2. Transfer to agarose coated dish containing ¾ NAM (make fresh)

NAM 100mL:

7.5 ml 10X NAM salts

2 ml phosphate buffer

0.75 ml bicarb (0.84 g in 100 ml H20)

89.75 ml H20

  1. Cut 10 – 20 caps per sample
  2. Wash caps and transfer to Eppendorf tube (USA Scientific)
  3. Culture to desired stage

Harvest Caps

  1. Add 20 µl Proteinase K to 1 mL Caps lysis buffer    Caps Lysis Buffer  -   for 100mL      
  2. Remove NAM and loose particles                                0.5% SDS           -  2.5 mL (20%)
  3. Add 100 µl PK mix (200 µl for whole embryo)             5 mM EDTA        -  1 mL (500mM)
  4. Pipette up and down to homogenize                          50 mM Tris pH7.5 -  5 mL  (1M)
  5. Incubate at 42°C for 30 minutes                                  50 mM NaCl          -  1 mL  (5M)  
  6. Freeze or continue to RNA isolation                                   H20                 -  90.5mL

RNA Isolation

  1. Add equal volume of Phenol/Chloroform, vortex and spin for 3 minutes
  2. Save top layer (100 µl for caps, 200 µl for whole embryo)

  3. Transfer to tube containing:

    1. 10 µl 10M NH4OAc or 3M NaOAc (20 µl whole embryo)

    2. 1 µl glycogen (2 µl whole embryo)

    3. 250 µl ETOH (500 µl whole embryo)

      4.
  4. Spin 10 minutes, discard supernatant, wash with 70% ETOH, spin 10 minutes, dry, re-suspend in 15 µl of DEPC H20
  5. Freeze or continue

 

DNAse Treatment

    Ingredient                    µl/reaction

    10X DNAse Buffer       2.5

    DNAse 1                       0.4

    20mM DTT                   1.25

    RNAse Inhibitor            0.5

    H20                                5.4

    total                             10.05

  1. Add 10.05 µl of mix to each sample
  2. Incubate at 37°C for 30 minutes
  3. Add 75 µl H20
  4. Add 100 µl Phenol/Chloroform, vortex and spin 3 minutes
  5. Save 100 µl top layer, transfer to new tube containing:
  6. 10 µl 10M NH4OAc or 3M NaOAc
  7. 250 µl ETOH
    8.
  8. Spin 10 minutes, discard supernatant, wash with 70% ETOH, spin 10 minutes and dry

Reverse Transcription
  1. Re-suspend pellets in 10 µl H20 + 1 µl hexamers (0.1 mg/ml)
  2. (20 µl H20 + 2 µl hexamers for whole embryo)
  3. 65°C for 4 minutes, cool on ice
  4. Make up the following mix;

Ingredient                       µl/reaction

5X first strand buffer         4

20 mM DTT                       1

RNAse inhibitor                0.5

dNTP’s (10mM)                1

H20                                     2.25

  1. Get an empty tube and label it RT- then add 9 µl of mix + 11 µl whole embryo, set aside
  2. Add reverse transcriptase to mix (0.25 µl/reaction)

  3. Add 9 µl of  mix to each sample, including the REMAINING 11 µl of whole embryo*

  4. 42°C for 30 minutes

  5. Freeze or continue

*here you should have 2 whole embryo tubes containing embryo

and mix, but only one having the reverse transcriptase added to it

 

 

 

 

PCR

  1. Set up PCR using the following mix;

Ingredient                       µl/reaction

10X PCR buffer                 5

MgCl2                                 4

dNTP’s (10mM)                1

Taq                                     0.5

Primer 1                             1

Primer 2                             1

cDNA                                 1

H2O                                  34.5

Total                                  48*

 

*we know this should add to 50µl, but it works this way

  1. Run PCR with appropriate cycle settings

  2. Run PCR products on gel