Main Page: Difference between revisions

@@ Line 20: / Line 20: @@
 ===[[Digest and Ligation]]===
 ===[[Gel Purification]]===
-Using the NEB #T1020 Monarch Kit
-For a detailed protocol, or to download the full manual, visit www.neb.com/T1020
-BEFORE YOU BEGIN:
-*Add 4 volumes of ethanol (2 95%) to one volume of DNA Wash Buffer. (Needed to do the first time that the kit is used).
-*All centrifugation steps should be carried out at 16,000 xg (-13,000 RPM).
-*Please note: column holds 800 ul.
-PROTOCOL STEPS:
-# Run your digested DNA on an agarose gel. Use the 'wide combs' that will allow for more volume inside of them. Run the gel slowly (40-60 volts) for better band separation.
-# Weigh empty micro-centrifuge tubes, and write the weight on the outside of the tube.
-# Using the UV illuminator in Exx (across from Superlab), excise the DNA fragment from the agarose gel, taking care to trim excess agarose. Transfer to the 1.5 ml micro-centrifuge tube. Minimize exposure to UV light. Weigh the gel slice + micro-centrifuge tube and calculate the weight of just the agarose chunk. Agarose in a tube can be frozen at -20 and extracted later — this is a potential stopping point in the protocol.
-# Turn on the small water bath and set to 50°C. Put in the DNA Elution Buffer bottle, ensuring that it does not tip over in the water bath.
-# Add 4 volumes of Gel Dissolving Buffer to the gel slice (e.g., 400 ul buffer per 100 ul or 100 mg agarose).
-# Incubate the sample at 50°C, vortexing periodically until the gel slice is completely dissolved (generally 5-10 minutes). For DNA fragments > 8 kb, an additional 1.5 volumes of water should be added after the slice is dissolved to mitigate the tighter binding of larger pieces of DNA (e.g., 100 ul gel slice: 400 ul Gel Dissolving Buffer: 150ul water).
-# Insert column into collection tube and load sample onto the column with the mixture. Remember that the column can hold only 800ul maximum; you may need to load 800ul; spin; discard flow-through; load remaining liquid; spin again. Spin for 1 minute, then discard flow-through.
-# Re-insert column into collection tube. Add 200 ul DNA Wash Buffer and spin for 1 minute. Discarding flow-through.
-# Repeat step 5 by adding 200 ul DNA Wash Buffer and spin for 1 minute.
-# Transfer column to a clean 1.5 ml micro-centrifuge tube. Use care to ensure that the tip of the column does not come into contact with the flow-through. If in doubt, re-spin for 1 minute.
-# Add 6ul - 20ul of DNA Elution Buffer at 50°C to the center of the matrix. Wait for 1 minute, and spin for 1 minute to elute DNA. Molecular grade water (pH 7-8.5) can also be used to elute the DNA. Yield may slightly increase if a larger volume of DNA Elution Buffer is used, but the DNA will be less concentrated.
 ===[[Creating Competent E. coli Cells]]===

Revision as of 17:05, 30 August 2021

General microbiology protocols

Cloning and gene manipulation

Arabidopsis thaliana protocols

Streptococcus pneumoniae protocols

Streptococcus suis protocols

Interesting Podcats to Listen to When Doing Lab Work!

Cambridge protocols

Getting started with MediaWiki

Navigation menu

Search