IGEM:Harvard/2009/General Protocols
QIAprep Spin Miniprep Kit
This protocol is designed for the purification of up to 20 ug high-copy plasmid DNA from 1-5 ml overnight E. coli culture in LB medium.
Procedure
- Resuspend pelleted bacterial cells in 250uL Buffer P1 and transfer to a microcentrifuge tube.
- Add 250 uL Buffer P2 and mix thoroughly by inverting the tube 4-6 times.
- Add 350 uL Buffer N3 and mix immediately and thoroughly by inverting the tube 4-6 times.
- Centrifuge for 10 min at 13,000 rpm in a table-top microcentrifuge.
- Apply the supernatant to the QIAprep spin column by pipetting
- Centrifuge for 30-60 s. Discard flow-through.
- Wash QIAprep spin column by adding 0.75 ml BUffer PE and centrifuging for 30-60s.
- Discard the flow-through, and centrifuge for an additional 1 min to remove residual wash buffer.
- To elute DNA, place the QIAprep column in a clean 1.5 ml microcentrifuge tube. Add 50 uL Buffer EB to the center of each QIAprep, let stand for 1 min, and centrifuge for 1 min.
Restriction Digest, using New England Biolabs (NEB) Enzymes
- Pick a volume where it will be easy to calculate the quantities of 10x and 100x buffers to add and write out a quick recipe (See samples).
(You can add a little water or EB buffer to make this easy, without hurting the reaction)
- Pipette the DNA to cut into a 1.5ml eppendorf tube.
(Keep a few micoliters to run on a gel against the product, as a negative control)
- Add water if you need (See example below)
- Add 10X reaction buffer
(See the enzyme(s) page in the NEB manual or website for the correct one)
- Add 100X BSA if needed
(BSA is recommended for some reactions (see manual), and will not hurt any)
- Add restriction enzyme(s) last
(Keep these on ice or in a freezer box, Always) (Also, keep the percentage of enzyme in the reaction below 5% by volume to avoid nonspecific cutting)
- Let the reaction run at the temperature recommended in the NEB manual for at least 2 hours.
(Overnight is ok)
- Run a bit of the digest sample against the undigested control to confirm that the digstion worked.
Sample single digest (50 ul total volume)
- 30ul DNA
- 13.5ul Water
- 5ul 10X Buffer
- 0.5ul 100X BSA
- 1ul Restriction Enzyme
Sample double digest (50 ul total volume)
First, check the NEB manual double digest page for optimal conditions or whether it is recommended against for your enzymes
- 30ul DNA
- 12.5ul Water
- 5ul 10X Buffer (Check NEB double digest table - many not be what is used for the single digests
- 0.5ul 100X BSA (Add if it is required for either enzyme)
- 1ul Restriction Enzyme 1
- 1ul Restriction Enzyme 2