(for screening of E. coli colonies from cloning operations)
1. Prepare 18 X 150 mm test tubes with 5ml LB medium
2. Add appropriate amount of selective antibiotic to each tube
3. Prepare a plate of LB medium containing the same antibiotic selection.
4. Label the plate with numbers (you will use them to mark the location of the colonies you will spot onto the plate)
5. Pick colonies from the plate from your transformation reaction. (This is done by touching an individual colony with a toothpick or loop)
6. Once you have picked a colony from the plate, poke it on top (or touch gently if you are using a loop) of the first number of your new plate. Then place the toothpick in the test tube with LB or swirl your loop around in the LB.
7. Proceed to the next colony using a new toothpick each time or flaming the loop between colonies. Be careful to allow the loop to cool before touching the next colony.
Note that picking colonies can be performed on a bench top since all work is done using antibiotic selection.
8. Grow the tubes in a 37C shaker at 200RPM overnight (about 16Hr)
9. Next day the tubes should be well grown and very cloudy. Discard any that are not.
1. Get all of your reagents ready. Label 2 sets of 1.5ml epitubes (two for each test tube) Make them differentiated by using 2 colors of pen. Put 0.4% SDS in water bath if precipitated.
2. Collect the cells by carefully decanting into 1.5ml epitubes (Try to fill them but leave some room for the cap to close. Volumes do not need to be exact.)
3. Spin the cells down for one minute at maximum speed in a bench top micro centrifuge.
4. Remove from the centrifuge and decant or aspirate off the supernatant. Should have a nice pellet of cells in the bottom and supernatant should be clear. Be sure to get as much of the remaining liquid out as possible. Blot dry if decanting.
5. Resuspend the pellet by vortexing in 100ul of Solution I. There should be no chunks of pellet remaining.
6. Make Solution II from 50% 0.4M NaOH and 50% 0.4% SDS. Add 200ul to each epitube. Use a P1000 because SDS can be easily sucked into smaller pipets and cause damage. (If you are careful you can try to use only one tip for the whole set of tubes as long as you don’t touch any of them)
7. Invert several times and incubate at room temperature for 5 minutes –this incubation may be skipped if you are in a hurry. You can start adding one ml of 98%-95% EtOH to the second set of tubes while you are waiting. DO NOT vortex at this stage
8. Immediately after the 5 minutes add 150ul of Solution III to the mix. Invert the tubes several times to mix. Incubate on ice 5 minutes. This incubation can be skipped .
Don’t vortex here either.
9. After 5 minutes is over, spin for 5 minutes at maximum speed in a bench top microcentrifuge. Invert tubes again before spinning.
10. Remove the tubes promptly from the centrifuge once the spin is done. Carefully remove 200ul of clear solution (Avoid chunks of pellet and pipet from near bottom of tube) and put it into the second tube, which should have 1ml of 98% -95% EtOH in it. Continue with each sample. And of course change tips between each one. Do NOT allow the solution to sit around. Once it is on EtOH though you can stop if necessary.
11. Invert the tubes and spin for 5 minutes at max speed as before.
12. After spinning you should have an obvious white colored pellet. This pellet contains DNA and some protein. Carefully decant the EtOH off into a container or down the drain. Be sure to blot the tubes dry. You want to get ALL of the EtOH out.
13. Dry the DNA by spinning in a centrivap for about 5 minutes or air dry for approximately 1 hour. The Pellet should not be overdried but in my experience even great dryness has not made it difficult to resuspend.
14. Resuspend the DNA in 20-50ul of TE + Rnase. I usually use 30ul.
1. Look over your construct’s map (you did make a map right?) and figure out what enzymes you would like to check you construct with. If you didn’t destroy the sites you used to clone the fragment (by compatible ends or blunting) you can just use those same enzymes. In any case you should select enzymes that will give you a diagnostic fragment of at least 200-300bp. Much smaller fragments become difficult to see on the gel and 500bp and up are best. Note that DNA produced by the above protocol is SALTY and dirty and some enzymes such as Sac I will not cut it well.
2. Cut the DNA for approximately 1 hr at 37C. Some enzymes are different however and you should always check the catalog before you do a digest. You may want to include a positive control to make sure your restriction worked.
3. Sample reaction:
2ul DNA
1ul BamHI Buffer
1ul BSA
0.1ul BamHI
0.1ul SpeI
5.8ul H2O
10ul total
4. After cutting, load all 10ul of your digest on a minigel with narrow wells. Use 0.8% Agarose and run for about 1 hr at 100Volts unless your insert is smaller than 300bp then you might want to increase agarose content to 1%.
5. Visualize the bands by staining with Ethidium Bromide. Look for your band of interest. Don’t forget to load a ladder.
50mM Glucose
25mM Tris-HCl (pH 8.0)
10mM EDTA (pH 8.0)
0.2% SDS
0.2 M NaOH
60mL 5M Potassium acetate
11.5mL Acetic Acid
28.5mL H2O