Berkeley Protocols
From 2006.igem.org
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| + | <h1>Protocols</h1> | ||
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| + | ==Innoculations== | ||
| + | |||
| + | 10mL of LB | ||
| + | 5microliters of 2000X Amp | ||
| + | 10 microliters of 1000X Chloramphenicol | ||
| + | 50 microliters of 200X Kan | ||
| + | |||
| + | Half everything if only growing for sequencing | ||
| + | |||
| + | ==Plasmid Prep== | ||
| + | |||
| + | For sequencing elute in 30microliters of H20 | ||
| + | For restriction elute in 40microliters of EB | ||
| + | Nanodrop and write concentration on tube with date | ||
| + | |||
| + | Restriction: | ||
| + | |||
| + | In PCR tube if running overnight 8hr at 37C | ||
| + | Can add everything to plasmid prep tube if digesting in 37degree water bath (6hr) | ||
| + | 5ml 10X BSA | ||
| + | 5ml NEB Buffer | ||
| + | 37ml of purified plasmid (should be this much after eluting and nanodropping) | ||
| + | 1.5mL of Enzyme 1 | ||
| + | 1.5mL of Enzyme 2 | ||
| + | |||
| + | Suffix insertion: | ||
| + | Insert: Spe I, Xba I, Buffer 3 | ||
| + | Vector: Spe I, Pst I, Buffer 2 | ||
| + | |||
| + | Prefix insertion: | ||
| + | Insert: EcoRI, Spe I, Buffer EcoRI | ||
| + | Vector: EcoRI, Xba I, Buffer 2 | ||
| + | |||
| + | Gel Purification: | ||
| + | |||
| + | Pouring the gel: | ||
| + | 25mL of 1X TAE | ||
| + | About 180mg of agarose powder | ||
| + | Swirl and cover with kimwipe before microwaving for about 45 seconds | ||
| + | Make sure all the agarose is dissolved before pouring into mold | ||
| + | Use taped off comb and longer mold | ||
| + | Once the surface of the mold is covered it should be enough volume | ||
| + | |||
| + | Loading and running the gel: | ||
| + | Write down the order of samples in the lab notebook | ||
| + | Spin down tubes briefly in the little centrifuge | ||
| + | Add 5microliters of loading dye to each tube. | ||
| + | Mix and load slowly into well should be 50-55microliters | ||
| + | Use one of the normal lanes for the ladder | ||
| + | Run at 90-110V for about 40 minutes | ||
| + | |||
| + | Excising the bands: | ||
| + | Get a new razor blade | ||
| + | Before putting the gel on the uv box make sure you have an idea of which lane is vector and which is insert as well as relative sizes. | ||
| + | Prepare 1.5ml tubes for each sample | ||
| + | Once you turn on the uv light be as quick as possible cutting the gel, do not take a picture first. | ||
| + | Put each gel slice in a 1.5ml tube | ||
| + | If you’re not sure if the fragments were the right size then take a picture afterwards, you can compare where you cut to the ladder | ||
| + | Blank the scale with an empty tube then weigh each sample. | ||
| + | Add 2x volume of buffer QG to each tube. 100mg gets 200microliters of QG | ||
| + | Dissolve in 55C water bath for 10minutes | ||
| + | Proceed with gel purification protocol | ||
| + | Elute in 30ml of EB and nanodrop | ||
| + | |||
| + | Ligations: | ||
| + | |||
| + | I usually use 50-100ng of vector which hopefully is about 2-3 microliters. | ||
| + | Then I add insert to a total of 8 microliters so if I used 2 of vector I use 6 of insert | ||
| + | Another tube is for vector only so use the same amount of vector as in the normal tube and add to 8microliters of H20 | ||
| + | Add 1 microliter of ligase buffer | ||
| + | Add 1 microliter of DNA ligase | ||
| + | Can put in fridge overnight or move on to transformation immediately | ||
| + | |||
| + | Transformation (chemical) | ||
| + | |||
| + | Make sure water level in 42 degree incubator is not too low otherwise add some | ||
| + | Get appropriate plates and put them in 37C incubator | ||
| + | Get ice bucket and take out the 5X KCM buffer and sterile water 50ml Falcon tubes from the fridge | ||
| + | Get enough tubes of chemically competent cells (hopefully we will have our own stock) | ||
| + | Each tube is enough for two ligations | ||
| + | Spin the tube down very briefly after thawing on ice and leave it on the ice | ||
| + | Add 70microliters of H20 to each ligation tube | ||
| + | Add 20microliters of 5X KCM | ||
| + | Finally add 100microliters of cells and put on ice for 20minutes (use timer) | ||
| + | After 20 minutes place in 42C water bath for 1.5 minutes | ||
| + | Remove and place back on ice | ||
| + | Plate the whole tube’s volume immediately | ||
Revision as of 09:23, 13 November 2005
Protocols
Innoculations
10mL of LB 5microliters of 2000X Amp 10 microliters of 1000X Chloramphenicol 50 microliters of 200X Kan
Half everything if only growing for sequencing
Plasmid Prep
For sequencing elute in 30microliters of H20 For restriction elute in 40microliters of EB Nanodrop and write concentration on tube with date
Restriction:
In PCR tube if running overnight 8hr at 37C Can add everything to plasmid prep tube if digesting in 37degree water bath (6hr) 5ml 10X BSA 5ml NEB Buffer 37ml of purified plasmid (should be this much after eluting and nanodropping) 1.5mL of Enzyme 1 1.5mL of Enzyme 2
Suffix insertion: Insert: Spe I, Xba I, Buffer 3 Vector: Spe I, Pst I, Buffer 2
Prefix insertion: Insert: EcoRI, Spe I, Buffer EcoRI Vector: EcoRI, Xba I, Buffer 2
Gel Purification:
Pouring the gel: 25mL of 1X TAE About 180mg of agarose powder Swirl and cover with kimwipe before microwaving for about 45 seconds Make sure all the agarose is dissolved before pouring into mold Use taped off comb and longer mold Once the surface of the mold is covered it should be enough volume
Loading and running the gel: Write down the order of samples in the lab notebook Spin down tubes briefly in the little centrifuge Add 5microliters of loading dye to each tube. Mix and load slowly into well should be 50-55microliters Use one of the normal lanes for the ladder Run at 90-110V for about 40 minutes
Excising the bands: Get a new razor blade Before putting the gel on the uv box make sure you have an idea of which lane is vector and which is insert as well as relative sizes. Prepare 1.5ml tubes for each sample Once you turn on the uv light be as quick as possible cutting the gel, do not take a picture first. Put each gel slice in a 1.5ml tube If you’re not sure if the fragments were the right size then take a picture afterwards, you can compare where you cut to the ladder Blank the scale with an empty tube then weigh each sample. Add 2x volume of buffer QG to each tube. 100mg gets 200microliters of QG Dissolve in 55C water bath for 10minutes Proceed with gel purification protocol Elute in 30ml of EB and nanodrop
Ligations:
I usually use 50-100ng of vector which hopefully is about 2-3 microliters. Then I add insert to a total of 8 microliters so if I used 2 of vector I use 6 of insert Another tube is for vector only so use the same amount of vector as in the normal tube and add to 8microliters of H20 Add 1 microliter of ligase buffer Add 1 microliter of DNA ligase Can put in fridge overnight or move on to transformation immediately
Transformation (chemical)
Make sure water level in 42 degree incubator is not too low otherwise add some Get appropriate plates and put them in 37C incubator Get ice bucket and take out the 5X KCM buffer and sterile water 50ml Falcon tubes from the fridge Get enough tubes of chemically competent cells (hopefully we will have our own stock) Each tube is enough for two ligations Spin the tube down very briefly after thawing on ice and leave it on the ice Add 70microliters of H20 to each ligation tube Add 20microliters of 5X KCM Finally add 100microliters of cells and put on ice for 20minutes (use timer) After 20 minutes place in 42C water bath for 1.5 minutes Remove and place back on ice Plate the whole tube’s volume immediately
