2. Amplify by PCR the desired segment of DNA. You generally will only use 14 ul of the PCR product, so you can get by with a 20-25 ul PCR reaction.
3. Run 5 ul PCR reaction on a 1.5% agarose gel to ascertain success of amplification.
4. Prepare a master mix to include: 1 ul
enzyme buffer (e.g. 10X NEBuffer2) + 2 units restriction enzyme (=0.25
Alu I but the number of ul will vary from enzyme to enzyme - look on the enzyme tube itself for information) per
sample. This is a miniscule amount of enzyme but diluting the enzyme in buffer for long term storage can dramatically decrease enzymatic activity over time. Just use accurate pipetters or add a little extra buffer and enzyme to your master mix to make sure you have enough mix for all of your samples. If BSA is included with enzyme, add 0.1 ul BSA (10 mg/mL) per sample to master mix.
5. Put appropriate amount of master mix into
each tube and add 9.0 ul PCR product. Place in 37 C incubator for
6. Run 6 ul of digested DNA through a 1.5%
agarose gel stained with ethidium bromide. You may want to run a
standard to size bands and/or a positive control to make sure the digestion worked.
1.0 *l 10X NEBuffer 4 X = *l Buffer
0.1 *l BSA (10 mg/mL) X = *l BSA
0.2 *l Nla III (2 units) X = *l Nla III
1.3 *l master mix per reaction
+9.0 *l PCR product (Marten 37 & MVZ 14)
americana group - 41 541 153 46
caurina group - 41 200 341 199
1.0 *l 10X NEBuffer 2 X = *l Buffer
0.25 *l Alu I (2 units) X = *l Alu I
1.25 *l master mix per reaction