Mini-Prep Protocol for Purification of Plasmid DNA from Bacteria

After growing up selected bacterial colonies overnight in 1.5 mls of LB broth, the bacteria can be lysed and the plasmid DNA separated from the larger chromosomal DNA.

1.  Mix the broth in the culture tube, then transfer 1.0 ml to an eppy tube.

2.  Pellet the cells by centrifugation for 1’ at 13,200 rpm in the microfuge.  Discard the supernatant carefully and completely then resuspend the cell pellet in 200ml of Solution A.  Let sit for 5’.

3.  Add 400ml of Lysis Solution B and mix by inverting the tube several times.  Mix gently!

4.  Add 300ml of Neutralization Solution C, which will immediately cause a white precipitate to form.  Mix by inverting the tube eight times then spin at 13,200 rpm for 6 minutes.

5.  Carefully save the supernatant to a new eppy tube and discard the tube with the leftover white precipitate.

6.  Add 500ml of isopropanol and mix well by vortexing briefly.

7.  Centrifuge for 10’ at 13,200 rpm.  Dump the supernatant carefully and wash pellet with 50 ml of 95% ethanol by adding then immediately removing the EtOH.

8.  Being careful that the pellet does not slide down the side of the tube, remove any obvious ethanol moisture from the tube with a scrap of Kimwipe (DO NOT TOUCH THE PELLET WITH THE KIMWIPE!).  Let the pellet dry for 5’ by setting the tube (lid open) on its side.  Resuspend the dried pellet in 50ml of dH₂O.

Mini-Prep Solutions:

Solution A:        25mM Tris-HCL, pH 7.5              (1.5 g Tris/500ml)

                        10mM EDTA                             (1.46 g EDTA/500ml)

                        50mM glucose                          (4.51 g Glucose/500ml)

Solution B:        0.2M NaOH (denatures chromosomal DNA)    (*1ml = 100 ml 10% SDS + 40 ml 5N NaOH + 860 ml dH₂O)

1% SDS (disrupts membranes, denatures proteins)

Solution C:        5M Potassium Acetate, pH 4.8 with Acetic Acid (restores neutral pH causing the chromosomal DNA, membranes and most proteins to precipitate, leaving soluble plasmid DNA and RNA in solution).

                        (60 ml 5M KOAc (49g/100ml) + 11.5 ml Glacial Acetic Acid + 28.5 ml dH₂O)

Digests of Mini-Prep DNA

8 ml DNA

1 ml Restriction Enzyme plus RNase

2 ml 10x Buffer

9 ml water

20 ml total          Incubate at 37C (unless indicated otherwise) for 45 minutes.

PCR reactions

?? ml Template DNA (1:100 dilution)

1.0 ml Primer 1 (1:100 dilution)

1.0 ml Primer 2 (1:100 dilution)

??  ml water

12.5 ml Green Demon Taq reagent

25 ml total

pGEM-T Ligations

Ratio of insert:vector should be approximately 3:1, with a total of 300 nanograms of DNA (.3 mg).  Usually the following reaction is appropriate:

3.0 ml PCR product

5.0 ml Ligation Buffer

1.0 ml vector

1.0 ml ml T4 DNA ligase

10.0 ml total      

Incubate at room temperature for 2 hours or overnight at 4 degrees C.  All or half of the ligation can be transformed.

Transformation of DH5a Competent Cells with Ligations

1.  Add 40ml of competent cells (in ice bucket) to your ligation reactions and replace them on ice.

2.  After 15’ on ice, place the tube at 37 degrees C for 90 seconds.

3.  After 90 seconds, quickly add 800ml of LB broth, cap the tube tightly, mix and then return to the 37 degree water bath for 35 minutes.

4.  Spin down the cells in the microfuge and withdraw 600ml of broth, resuspend the cells carefully in the remaining 200ml by tapping the tube gently.

5.  Spread 40ml of cells on the plate* and incubate (inverted) at 37 degrees C overnight.

*If using Blue/White screening, spread 15 ml of 40mg/ml X-Gal on plate prior to adding bacteria.

Agarose Electrophoresis

Agarose gels are defined by their percentage agarose in 1x TBE (Tris-Boric Acid-EDTA buffer).  A total volume of 30ml is sufficient for the small gel boxes we will most commonly use.  After weighing the agarose and adding the buffer, microwave the mixture for 1’ then cool in the 37 degree C waterbath until you can safely touch it without discomfort.

                        Agarose            1x TBE

1%    gel:            .3   g              30ml

1.2% gel:            .36 g               30ml

1.5% gel:            .45 g               30ml

2.0% gel:            .6   g              30ml

LB Broth and Agar plates

Mix all the ingredients well using a stir bar before autoclaving.  Allow agar to cool to 60 degrees C before adding ampicillin and pouring into plates.

                                                BactoTryptone              NaCl                 Yeast Extract                Difco Agar       

1.0 liter LB Broth                         10 g                            10 g                    5 g                               none

500 ml  LB Broth                          5 g                              5 g                   2.5 g                             none

250 ml  LB Broth                          2.5 g                          2.5 g                 1.25 g                            none

1.0 liter LB agar (50+ plates)        10 g                            10 g                    5 g                               15 g

500 ml  LB agar (25+ plates)         5 g                              5 g                   2.5 g                             7.5 g

250 ml  LB agar (10+ plates)         2.5 g                          2.5 g                 1.25 g                            3.75 g

Animal Tissue DNA Isolation

1.  Remove a section of animal tissue approximately the size of this letter: O

2.  Combine your tissue section with the other people in your group (4 people or less) in the mortar and add liquid nitrogen to freeze the tissue.  Grind the tissue carefully with the pestle until it is powdered.

3.  Use a small spatula to divide the tissue into eppy tubes, one for each person in the group. 

4.  Add 250 ml of 10% Chelex (MIX CHELEX WELL BEFORE PIPETTING!) to your eppy tube.  Be sure the tube is tightly capped, and wrap the top in parafilm.

5.  Boil for 12’ in a water bath.

6.  Remove tube from water bath and place on ice for 2 minutes.

7.  Spin the tube for 1’ at top speed in the microfuge.

8.  WITHOUT DISTURBING THE PELLET, transfer 5 ml of the supernatant to a clean PCR tube.

9.  Setting up the PCR reactions:

            5 ml DNA (already in tube from step #8)

            1 ml forward primer

            1 ml reverse primer

            3 ml dH₂O

            10 ml Green Demon Taq enzyme mix

            20 ml total

Human DNA Isolation

1.  Add 250 ml of 10% Chelex (MIX CHELEX WELL BEFORE PIPETTING!) into an eppy tube. 

2.  Scrape as many cheek cells as possible using the sterile swab and dislodge them into the chelex solution.  Be sure the tube is tightly capped, and wrap the top in parafilm.

3.  Boil for 10’ in a water bath.

4.  Remove tube from water bath and place on ice for 2 minutes.

5.  Spin the tube for 1’ at top speed in the microfuge.

6.  WITHOUT DISTURBING THE PELLET, transfer 5 ml of the supernatant to a clean PCR tube.

7.  Setting up the PCR reactions:

            5 ml Human DNA (already in tube from step #6)

            1 ml TPA-1 primer

            1 ml TPA-2 primer

            3 ml dH₂O

            10 ml Green Demon Taq enzyme mix

            20 ml total

PCR cycle parameters

     35 cycles of the following:

            Denaturation                 94 C     1min

            Annealing                      50 C     30 sec

            Extension                     72 C     1 min

Reverse Transcriptase PCR (RT-PCR)

This reaction requires sterile technique and the correct amount of RNA template to work effectively.  Each reaction should have 1 mg of RNA.

Experimental reaction:                                          Control reaction (1 per group):

            12.5                              RT-PCR 2x master mix   12.5

            ?                                  RNA template                            ?

            1.0                                Forward primer              1.0

            1.0                                Reverse primer              1.0

            0.5                                RTase Blend                             (Do not add)

            ?                                  Water    (up to 25)                      ?

It would be more accurate to mix a cocktail together for the experimental reaction, then divide it equally among the 4 members of your group:

            50, (do not add RNA to the cocktail), 4, 4, 2, (do not add water to the cocktail)

Each member will add their RNA sample and the appropriate amount of water to their PCR tube before adding the cocktail.

Here are the cycle parameters we will be using:

First strand synthesis:               47 C, 30 minutes

RTase inactivation/                    94 C, 2 minutes

Initial denaturation

Denaturation                             94 C, 20 seconds

Annealing                                  50 C, 30 seconds                      40 cycles

Extension                                 72 C, 1 minute

Final extension              72 C, 5 minutes

8% Polyacrylamide Gel Electrophoresis

11.5 ml dH₂O

4.5 ml 5x TBE

6.2 ml Acryl/Bis (29:1 = 29 g Acrylamide + 1 g Bis in 100 ml dH₂O)

200 ml 10% Ammonium persulfate (APS)

20 ml TEMED

10x TBE

108 g Tris

55 g Boric Acid

40 ml 0.5M EDTA  (0.5M EDTA = 190.1 grams/liter or 95 grams/500 ml)

1000x Stock Ampicillin

50 mg/ml concentration, prepare 25 ml.  Dissolve in water, added 10N NaOH to dissolve, then adjust pH to 7.5-8.0.  Filter sterilize with .22 mm filter.