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Mouse Breeding
Mice born within a week from each other can be considered as belonging to the same cohort
If genotyping PCR reactions are not successful, you might want to consider using more DNA (e.g. double the normal amount)
Mice are weaned at approx 3 weeks of ageAt 2 weeks you should genotype so that by the time you wean you will already know the genotype of each pup
The ones you don't need, you sac
The ones you need you wean into male and female cages (5 each/cage)
Pups cannot breed until 6-8 weeks old
At that time, you separate out the males and breed with up to 3 females per cage (harem mating)
Pregnant (plugged) females are separated from the males the next day
Isolation of mouse tail genomic DNA
1. Add 500uL of lysis buffer containing 300ug/mL Proteinase K.
2. Shake at 55C overnight.
3. Spin at max speed 5 minutes to pellet insoluble materials.
4. Remove supernatant to a clean tube.
5. Add 1/3 volume (approx. 170uL) 6M NaCl to supernatant.
6. Vortex lightly.
7. Spin at max speed 20 minutes to pellet proteins.
8. Remove supernatant to a clean tube.
9. Fill tube with supernatant with 100% ethanol (1ml).
10. Spin at max speed for 10 minutes.
11. Remove and discard supernatant.
12. Add approx. 1mL 70% ethanol.
13. Spin at max speed 20 minutes.
14. Remove and discard supernatant.
15. Allow pellet to dry 5 minutes.
16. Resuspend pelleted DNA in 100uL water or TE.
Mouse tail lysis buffer
| Component | Final Concetration | Volume |
| | ||
| 1M Tris-HCl (pH ~8.0) | 100 mM | 50 mL |
| 0.5 M EDTA | 5 mM | 5 mL |
| 10 % SDS Solution | 0.2 % | 10 mL |
| 5 M NaCl | 200 mM | 20 mL |
| dH2O | --- | 415 mL |
| | | |
| FINAL VOLUME: | | 500 mL |
Store at RT.
Proteinase K should be added immediately prior to use at a final concentration of 1 mg/mL (5 uL of 20mg/mL stock per 100 uL Lysis Buffer). Do not store once Proteinase K has been added.
Gitschier buffer for Mouse Tail Lysis - Recipe
| stock concentration | amount to use | ||
| 67 mM Tris-HCL (pH 8.8) | 1M | 33.5 | |
| 16.6 mM ammonium sulfate | 1M | 8.3 | |
| 6.5 mM MgCl2 | 1M | 3.25 | |
| 0.5% Triton X-100 | 20% | 12.5 | |
| 100% | 5 | |
| 100 µg/ml proteinase K | |||
| ddH2O | 437.45 |
| Add proteinase K and bME fresh | |||||||
| 1. Add 200ul of Gitschier buffer for around 1/2 cm tail. You can add 100ul of Gitschier buffer if you have smaller amount of tissue. | |||||||
| 2. Digest tissue at 55C o/n | |||||||
| 3. Heat samples at 94C for 5 minute to heat inactivate the proteinase K | |||||||
| 4. Vortex the samples for 2-5 minutes on high setting to shear the DNA | |||||||
Alternatively for steps 3&4, if you have an eppendorp tube shaker you set the heat to 94C | |||||||
| and shaking to 1400, and just let your samples incubate for 15 minutes | |||||||
| 5. Spin down the samples at 13-14K for 1-2 minutes | |||||||
| 6. Can transfer the supernatant to new tube then use or just take 2ul of DNA for PCR | |||||||
| *For each sample add 2ul of proteinaseK and 2ul of bME to 196ul of buffer | |||||||
Mouse Morphometry
Of course you should measure body length and width, since many pathologic conditions are accompanied by developmental abnormalities
Tibia and femur length is a better indicator for mouse development than total body weight/length, since the latter may be lower due to nutritional causes
Tibia and femur length is a better indicator for mouse development than total body weight/length, since the latter may be lower due to nutritional causes
Rat Pancreas Harvest Protocol
The rats will be euthanized by 100% CO2 inhalation. The ventral midline incision site will be prepared by clipping with #40 clippers, gentle scrubbing with Betadine, and swabbing with alcohol. This procedure will be repeated twice. A #10 blade and scalpel will be used to make a 10cm midline ventral incision extending from the manubrium to the mid-abdominal region.
Analyzing A Mouse Model
Measurements to take when analyzing mice: length, weight, nose-crown length, intra-ocular length
Measurements should be taken in triplicate
It is a good idea to normalize all measurements to body weight
Standard error calculator: http://www.rose-hulman.edu/~gahnjc/calculator.htm
Student's T-test calculator: http://studentsttest.com/
