Team:SDU-Denmark/Protocols/Restrictions

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Sadly, we had a lot of trouble with our restrictions, and this page will reflect that in including alot of different restriction protocols.

=Protocol 1=
 * 1) Pool plasmid and dry on vacuum centrifuge down to about 50uL
 * 2) Mix the following into one tube:
 * 3) 4uL Plasmid and RIP
 * 4) 5uL 10x Buffer
 * 0,5uL BSA
 * 1) Fill with water to 47uL (37,5uL)
 * 1,5uL Enzyme 1
 * 1,5uL Enzyme 2
 * 1) Incubate for 2 hours on 37 degress C.
 * 2) Inactivate the enzymes at 80 degress C for 20 minutes.
 * 3) Place product on -20 degress C or continue immediately to ligation.

=Protocol 2=
 * 1) Fast digest restriction enzymes. Fast digest restriction enzymes have proved more efficient for cutting DNA, and is less time-consuming to work with. Fast Digest enzymes can be bought at Fermentas.
 * 2) 24 ul water
 * 3) 2 ul enzyme
 * 4) 4 ul Fast Digest buffer
 * 5) 10 ul PCR product
 * 6) Leave for 15 min at 37 degrees. Afterwards, inactivate the enzyme for 20 min at 80 degrees.

Be aware, only to cut with ONE enzyme at a time.

After cutting with enzyme 1, isolate and purify the DNA fragments on a gel before applying enzyme 2.

The final volume when cutting is 40 ul.


 * 1) Add 4 ul loading buffer to the eppendorf tube and load directly onto gel.
 * 2) Run gel and purify from gel.
 * 3) Eluate with 10 ul when purifying from gel.
 * 4) Eventually, place your sample in the vacuum centrifuge in order to get a smaller volume and greater concentration before ligation is applied. Final volume prior to ligation should be 5 ul.