In silico Double Digestion
Simulation of Subtracted Restriction
experiment will be simulate:
- Both endonucleases will be palindromic
- Recognition sequence has no degenerated nucleotides
- One endonuclease cleaves genome very frequently and the
- One restriction enzyme (RE1) allows a filling reaction
to be performed with digoxigenin labelled Uridine (Dig-dUTP) and the
other restriction enzyme
allows a filling reaction with biotinylated Cytosine (Bio-dCTP).
- From the four types of fragments yielded after digestion,
RE2-RE1 and RE2-RE2 fragments (labelled in one or both ends with
are captured by streptavidin-coated magnetic beads (subtracted).
- The remaining RE1-RE1 fragments (which are digoxigenin
labelled) will be separated
by electrophoresis, transferred by Southern blot and band
When filling the form (see image bellow)...
- we will check the checkbox for "SRF", and the correct
fragment type will be selected automatically ,
- in this example we will select restriction enzymes MunI and TaqI from the lists of commercially
palindromic restriction enzymes,
- although in the example the checkbox "For
non-palindromic endonuclease discern complementary" is checked,
unchecking it makes no difference
in this case (due to palindromic nature of the selected endonucleases),
- selective nucleotides are not included in the experiment
method does not require them).
After clicking the "Amplify" buttom we will get 83 bands (see result here
In the example bellow the same experiment is performed, but in this
recognition sequence is introduced in the form.