35delG Mutation Detection by Bsl1 Enzyme Digestion
Do PCR using the special primers for the detection of the 35delG mutation
Forward: GGT GAG GTT GTGTAA GAG TTG G
Reverse: CTG GTG GAG TGT TTG TTC CCA C
PCR Protocol
Per 25 l Reaction for one sample
 PCRMix
15 l
 25M Primer F
1 l
 25M Primer R
1 l
 Sample DNA
1 l1
 D.D. H2O
12 l
We multiply each portion by the number of samples that we are doing +1 ( for
control).
Usualy 1l DNA is sufficient. If the reaction doesn’t work, try to change DNA quantity to reach
optimal concentration (100-300ng per reaction). Change water amount respectively.
1
PCR Program





94 0C  3 minutes
94 0C  1min
60 0C  1min
72 0C  1min
72 0C  5 Minutes
 END
35 times
Enzymatic digestion with Bsl1
Bsl1:
10X Buffers 3:
D.D. H2O:
PCR product
0.5 l
2.0 l
7.5 l
10.0 l
We multiply each portion by the number of samples that we are doing.
 Add a known heterozygote to the digestion reaction, for control.
 Tubes are incubated at 55 oC for 16 hours.
 We load 10 l of digested reaction on a 4% agarose gel.
Expected Fragments
Wildtype
Heterozygote
35delG Homozygote
207
207
181
181
26
26