Protocols for alkaline lysis mini-plasmid preparation

Alkaline lysis mini-plasmid preparation

You are provided with two cultures : one containing E. coli which have been transformed by the pGEM-T Easy vector containing the gene for the polobox domain, and one which contains bacteria which have not been transformed./p> QIAprep Spin DNA Purification System (QIAGEN)
Production of Cleared Lysate

• Transfer  1000µL of each culture into separate labeled Eppendorf tubes and centrifuge at 8,000rpm for 5 minutes.

• Remove supernatant from each of the tubes and resuspend in 250µL Buffer P1. Ensure that there are no cell clumps visible after resuspension of the pellet.

• Add 250µL Buffer P2 to each sample and invert 4-6 times to mix. The solution should turn blue as the cells are lysed.

• Add 350µL Buffer N3 to each sample and invert 4-6 times to mix. The blue colour should disappear and the solution should become cloudy as proteins precipitate.

• Centrifuge both tubes at 13,000rpm for 10 minutes at room temperature.

Binding of plasmid DNA

• Insert a labelled QIAprep spin column into its centrifuge tube for each sample.

• Transfer the supernatant from the lysis stage into each spin column. Be very careful not to disturb the pellet. If traces of the precipitated proteins are present in the supernatant, re-centrifuge the tubes.

• Centrifuge the spin columns at 13,000rpm for 1 minute at room temperature.

• Discard the flow through in each centrifuge tube and reinsert the spin columns.

Washing

• Add 500µL Buffer PB to each spin column.

• Centrifuge at 13,000rpm for 1 minute.

• Discard the flow through from each centrifuge tube and reinsert the spin columns.

• Repeat wash steps above with 750µL wash solution.

• Without adding any more wash solution, centrifuge the tubes dry at 13,000rpm for 5 minutes at room temperature.

Elution

• Label a sterile Eppendorf tube for each sample.

• Transfer each spin column to the labelled Eppendorf tube, taking care not to transfer any of the wash solution.

• Add 20µL Buffer EB to the spin column.

• Incubate at room temperature for 1 minute.

• Centrifuge at 13,000rpm for 2 minutes at room temperature.

• Discard the column and retain the eluate in the bottom of the Eppendorf tubes. Samples can be stored at -20°C or below until needed.

Restriction digest

Our first procedure will be to prepare restriction digests of each of our mini-preps. We will perform a restriction digest on the mini-prep from the untransformed cultures, even though they should not have any plasmid DNA. This is called a negative control (ie. it shows us what the results of the experiment should look like if it didn’t work.

Restriction digests require very small amounts of reagents to be added. Since this exercise is a check of our samples and we do not want to use all of them up, we will work with the barest minimum volumes.

The reagents required for our digest are :

• DNA Sample – generally added in the same proportion as the restriction enzyme

• Restriction Enzyme – generally added at 10% of the final volume

• 10X Buffer – a solution of salts which maintain the correct pH for the enzyme to function. “10X” refers to the fact that it is ten times more concentrated than it needs to be and so must be diluted by the addition of the other reagents. The volume of buffer used must be 10% of the final volume. There may be different buffers required for each restriction enzyme

• Water – used to make up the reaction mixture to the final volume

We will be performing an EcoRI digest on our sample to drop out the polobox inserts.

Use the following table to deliver the volumes needed for our restriction digest :

•  Add the components listed above to a labelled Eppendorf tube

• Incubate the tubes at 37°C for 1-2 hours

Electrophoresis

In order to check for the presence of the plasmid, we need to examine the size of the DNA fragments which result from our restriction digest. We do this using agarose gel electrophoresis.

Preparation of TAE buffer

Electrophoresis uses an electric field to “push” DNA fragments through the gel. To ensure that this occurs efficiently, all of the DNA must have a negative charge (to ensure that it is pushed away from the negative terminal). This is done using a buffer which keeps the experiment at a pH where all of the DNA is negatively charged.

The buffer most commonly used in DNA analysis is TAE, which stands for Tris – Acetate – EDTA (where EDTA stands for ethylenediaminetetracetic acid). The buffer is usually made up at 50X concentration and then must be diluted when needed (this allows us to make and store large amounts of the buffer without having to remake it as often).

For your group, you will only need around 500mL of TAE buffer. You will need to prepare 500mL of 1X TAE buffer from the 50X stock solution provided. Perform the following calculations :

Total volume     = 500mL

1/50 of 500mL     = 500 ÷ 50  =  ____ mL

Volume of 50X stock needed is ____ mL

Volume dH2O needed     = Total volume  - Volume stock needed 

= 500mL – ____ mL

= ____ ml

Dilute ____ mL stock in ____ mL of dH2O

• Use the calculations above to prepare 500mL of 1X TAE buffer

Preparation of gel

The gel used to studying DNA is made from agarose, a jelly-like substance derived from seaweed. This material is supplied in powder form, and must be dissolved in the TAE buffer. For our experiment, we require a gel containing 0.8% agarose, ie. 0.8g of agarose powder dissolved in 100mL of  buffer.

• Weigh out 0.8g of agarose powder and suspend in 100mL of TAE buffer in a conical flask. One quantity is sufficient for the entire class

• Microwave the solution on HIGH for 2 minutes (for a small gel).  Make sure that the agarose is completely dissolved by swirling the heated mixture roughly every 30 seconds.  Allow it to cool for 3 minutes.

• Wipe a plastic gel tray and comb with 70% ethanol and place in the electrophoresis tank so that the rubber tubing forms a seal with the sides of the tank.
• Add 8µL of SYBR-Safe into the melted agarose and swirl to mix. This substance is a dye which binds to the DNA and glows green under ultraviolet light – it allows us to see where the DNA has migrated in the gel.
• Pour the melted agarose into the gel tray.  Place the comb into the right position and allow it to set for approximately one hour (this can be done faster by placing the gel tray in the refrigerator.
• Carefully remove the comb from the gel.  Rotate the gel tray so that the wells are toward the negative (black) terminals (the top of the tank, assuming that the electrodes are on the right hand side).  Cover the gel with 1X TAE running buffer.

Loading the gel

The samples must now be loaded into the wells in the gel left by the comb. To make this process easier, we mix the samples with a blue dye and glycerol. The dye migrates before all of the DNA and we can use this to tell when to stop running the gel. The glycerol increases the density of the sample so that it sinks to the bottom of the well on loading. The dye is provided at 6X the required concentration. This means that we have to add it to the sample in a proportion which dilutes it 1 in 6 (ie. five times as much sample as dye). Use the following calculation to find out how much dye is needed to add to a given volume of sample :

We are going to use all 10µL of our digest product
 
If the volume of dye added is “x” 

x  +  Volume of DNA  =  6x

Volume of dye needed to add to 10µL of digest product =  _______  µL

• Prepare loading solutions for each of your samples and DNA ladder.

• Load all of the loading solutions into separate wells in the gel (loading the DNA ladder last into a separate well on the left or right hand side of your gel).  Make sure you write down where your sample is in the gel.

Running the gel

• Run the gel at 80V. There must be small bubbles rising from both ends of the electrophoresis chamber. Check after 5 minutes to make sure the gel is running (i.e. the dye front has moved, is relatively straight and has run the correct direction). Then allow the gel to run for the necessary amount of time (about 1 hour however, check that the dye front has almost run through the gel).

• Switch off the power pack and take the gel to the illuminator. Take a photograph, print off and glue into your workbook in the space below. Annotate the photograph using the ID table you completed above, indicating bands of interest.

• Pour away the buffer from the electrophoresis tank and rinse well with water. Rinse the gel tray and comb as well.

Interpreting your gel

Whenever we run a gel, we should always include a DNA “Ladder” which contains fragments of DNA of known size. This ladder serves as a reference point to indicate the size of the DNA fragments in our sample. A map of the ladder we are using in this exercise is provided in the figure to the right.

Examine the photograph of your gel and check the sizes of bands. If the EcoRI digest as successful, you should see a band at around 3kb representing the vector, and another at around 0.8kb for the insert. Of course, for the mini-preps derived from the untransformed controls, you should see no bands at all.