From 2006.igem.org

Revision as of 03:14, 17 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

Sequencing

In a 1.5mL tube add 700-1000ng of purified plasmid (at least 500ng) or >100ng of purified PCR product (must be in H20)
Add water to a total volume of 12 microliters
Add 1 microliter of appropriate sequencing primer
BBfwd and BBrev at 3.2pmol/microliter are in tubes in the freezer

Restriction Digest

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 (check needed buffer here:[http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/double_digests.asp])
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, NEB Buffer 3
Vector: Spe I, Pst I, NEB 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 (3-4 large lanes, 2-3 small ones) 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. (note: samples should weigh less than 200 mg for good yields)
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

Colony PCR

(perform on ice) Create colony suspensions:
select a colony and suspend in 20 microliters of water
streak a plate to check that you actually took a colony
-The mix:
11.4 microliters of nanopure H20
0.4 microliters of forward primer
0.4 microliters of reverse primer
0.4 microliters of dNTPs (in the door of Jonathan's fridge, purple box, tubes labeled "10" or "5")
2 microliters of Thermopol buffer (common -20 fridge)
5 microliters of colony suspension
0.4 microliters of Platinum Taq
(total volume of 20 microliters)
- put in thermocycler and run igemcolPCR or enter the cycles in manually as:
95C for 5:00 (mins)
95C for :30
47C for :30
72C for 1:15
72C for 10:00
(the preceding sequence x35)
4C for ever

Making Media

200X Kan:

• 67.5mg dry Kan (in stockroom) in 5mL nanopure H20