IGEM:Harvard/2009/General Protocols: Difference between revisions

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(New page: ==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=== #...)
 
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#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.
#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.
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==Restriction Digest, using [http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/default.asp 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 [http://www.neb.com/nebecomm/DoubleDigestCalculator.asp 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

Revision as of 20:37, 12 June 2009

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

  1. Resuspend pelleted bacterial cells in 250uL Buffer P1 and transfer to a microcentrifuge tube.
  2. Add 250 uL Buffer P2 and mix thoroughly by inverting the tube 4-6 times.
  3. Add 350 uL Buffer N3 and mix immediately and thoroughly by inverting the tube 4-6 times.
  4. Centrifuge for 10 min at 13,000 rpm in a table-top microcentrifuge.
  5. Apply the supernatant to the QIAprep spin column by pipetting
  6. Centrifuge for 30-60 s. Discard flow-through.
  7. Wash QIAprep spin column by adding 0.75 ml BUffer PE and centrifuging for 30-60s.
  8. Discard the flow-through, and centrifuge for an additional 1 min to remove residual wash buffer.
  9. 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)

  1. 30ul DNA
  2. 13.5ul Water
  3. 5ul 10X Buffer
  4. 0.5ul 100X BSA
  5. 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

  1. 30ul DNA
  2. 12.5ul Water
  3. 5ul 10X Buffer (Check NEB double digest table - many not be what is used for the single digests
  4. 0.5ul 100X BSA (Add if it is required for either enzyme)
  5. 1ul Restriction Enzyme 1
  6. 1ul Restriction Enzyme 2
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