Handout 9/21/2015
During step 4 setup your restriction digestion tubes (see other side), add the purified plasmids after
completion of step 10. LABEL YOUR TUBES WELL (group number, sample 1 through 4). Store remainder of
purified plasmids in freezer box.
INSERT CHECK BY RESTRICTION DIGEST
SET UP restriction digestion for insert check:
Label your tubes, group number, and 1 - 4 (e.g 1-1; 1-2; 1-3; 1-4)
Combine in a master mix:
4 microliter of EcoRI (storage buffers of restriction enzymes can inhibit reaction if >10% of final reaction volume)
6 microliter of 10x buffer
42 microliter of milliQ water, mix, aliquot 13 microliter to 4 tubes each.
Add 2 microliter of plasmid sample
Incubate 30 minutes, 37C.
Run 1% agarose gel
Inspect for presence absence of plasmids, inserts.
Home work (also if you did not run the gel)
1)
2)
3)
describe the basic functioning of the DEAE (Qiagen) method for purification of DNA.
Describe what banding pattern on the gel shows that the restriction digestion confirms the presence of a
plasmid with an insert.
Can you predict how many gel bands will result from the insert after restriction digest with EcoRI? Why not?
QIAprep Spin