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 age

At 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 concentrationamount to use
67 mM Tris-HCL (pH 8.8)1M33.5
16.6 mM ammonium sulfate1M8.3
6.5 mM MgCl21M3.25
0.5% Triton X-10020%12.5
{beta}
1% -mercaptoethanol
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

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.


The ribs will be transected bilaterally along the mid-clavicular line and the ventral aspect of the rib cage will be raised to reveal the liver. The common bile duct will be identified on the dorsal aspect of the liver and will be cannulated with a sterile polyethylene tube. A balanced salt solution will subsequently be infused into the pancreas via the tube in order to distend it. The pancreas will be identified and harvested. Our aim is to isolate the pancreatic islets using the collagenase digestion method of Lacy and Kostianovsky.

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/

Saturday, January 30, 2010

Molecular Biology, DNA, cells, Cell Biology, PCR, biology, protocols, molecular biology, research, protein, Protocols, Protein, RNA, Current Protocols, Methods in Molecular Biology, CSH Protocols, Science, Cell, Research, Laboratory