Testing PCR primers or Southern blot conditions for transgene detection

Probe or PCR testing prior to submission:

We can provide normal mouse DNA if you don't have any

Please submit a photocopy of the results.

Include hybridization and wash conditions for blots

Include PCR reaction and cycling conditions for PCR

Southern blot

Probe identity

hybridization solution

hybridization temperature

wash solution

wash temperature

Controls should include:

1. Normal Mouse DNA (NM) 5-10 micrograms

2. NM + 1 gene copy/cell equivalent of the gene construct fragment (see below)

3. NM + 5 gene copy/cell

4. NM + 10 gene copy/cell equivalent of the gene construct fragment

5. 1 gene copy/cell equivalent of the gene construct fragment alone

6. 5 gene copy/cell equivalent of the gene construct fragment alone

PCR

5' primer 3' primer

denaturing temp denaturing time

annealing temp annealing time

extension temp extension time # of cycles

Controls should include:

1. Normal Mouse DNA (NM) 1 microgram

2. NM + 1 gene copy/cell equivalent of the gene construct fragment

3. NM + 5 gene copy/cell

4. NM + 10 gene copy/cell equivalent of the gene construct fragment

5. 1 gene copy/cell equivalent of the gene construct fragment alone

6. 5 gene copy/cell equivalent of the gene construct fragment alone

7. DNA from a donor of the same species as the gene construct fragment (if applicable)

See example of calculation for copy standards for PCR

See example of calculation for copy standards for Southern blot