Miniprep Isolation of DNA

Each group should have a total of 6 each overnight cultures (3 for GFP and 3 for HA). It
is advised that half of the group prep the GFP samples and the other half of the group
prep the HA samples. You will be isolating plasmid DNA from 4 ml of your overnight
cultures. In order to save time, you will be spinning down 2 each 2ml samples of the
same culture.

1. Label 2 each 2 ml tubes for each of the cultures making sure that they are clearly
labeled with your group#/construct#/culture#. Vortex the overnight sample gently
and using a pipetman transfer 2 ml of your broth culture of transformed Escherichia
coli to the microcentrifuge tube.

2. Centrifuge for 3 minutes (microcentrifuge) at room temperature with the hinge of

the microcentrifuge tube toward the outside of the rotor.

3. Pour off the supernatant.

4. Resuspend the bacterial pellet in 125 l of Buffer P1 (detergent). Use the pipet tip
to “stir” the pellet as you gently pipet up and down. No clumps of cells should be
visible after resuspension of the pellet. Combine the two tubes from the same
culture. Each sample should now be in one tube (250 l).

5. Add 250 l of Buffer P2 (alkali) and mix thoroughly by inverting the tube 5 times.
Adequate mixing will result in a homogeneously blue colored solution.

6. Add 350 l of Buffer N3 (neutralizing solution) and mix thoroughly by inverting the
tube 5 times. Adequate mixing will result in a homogeneously colorless/white
solution.
7. Centrifuge for 10 minutes at room temperature. During this step, label (with the
culture tube identity) QIAprep spin columns and place them into empty collection
tubes.

8. Following centrifugation, pour the supernatant into a labeled QIAprep spin column.

9. Centrifuge the column for 1 minute at room temperature. The plasmid DNA will
"stick" to the column.

10. Remove the column from the collection tube and pour off the flow through.

11. Wash the column by adding 500 l of Buffer PB and centrifuging for 1 minute.

12. Remove the column from the collection tube and pour off the flow through.

13. Wash the column by adding 750 l of Buffer PE and centrifuging for 1 minute.

14. Transfer the column to a microcentrifuge tube and centrifuge for 1 minute to remove
any residual wash buffer.

15. Transfer the column to a second microcentrifuge tube and centrifuge for 1 minute to
remove any residual wash buffer from the column.

16. Place the column in a clean microcentrifuge tube (label this tube with the identity of
the clone). Add 75 l of water to the center of the column, let stand 5 minutes, and
centrifuge for 1 minute.

This becomes your stock tube of DNA that is used for the next set of experiments:
restriction digest, determination of concentration, DNA sequencing and
transfection. Nothing should ever be added to this tube as it represents the "final"
product of your experiment.
Restriction Digest

You will be digesting each plasmid sample with two restriction enzymes.

You will be digesting 200-500 ng (estimated yield) of plasmid in a single 10 l reaction.

1. Label 6 microcentrifuge tubes (3 each for GFP and 3 each for HA).

2. First, add 9 l of water to each tube. Next, add 1 l of plasmid to the appropriately
labeled tube.

3. Once you have set up all 6 tubes, you will need to go to the TA bench and get an
aliquot of restriction enzyme (see list on last page). Label three tubes for your
assigned enzymes. Go to the TA bench and get 35 l of EcoRI and 20 l of the
other two enzymes.

4. Once you have aliquots of the enzymes, return to your bench and add 5 l of each
enzyme to the 10 l of DNA/water (total = 20 l). Mix gently by pipeting up and
down two times.

5. Place your digest tubes at 37C for at least 30 minutes.

6. During the incubation you should pour the agarose gel that will be used to identify
the PCR products.

 Tape the end of the gel-forming tray

 Add 1 comb to the gel-forming tray
 Weight out 0.8 grams of agarose and add to Erlenmeyer flask
 Add 80 mls of TAE buffer
 Microwave
 Place the flask into the Microwave Oven and set the time for one minute. Watch
the solution and when it starts to boil stop the microwave. Do not boil it too long
or it will boil over. Use a pair of insulated gloves to remove the flask. Gently
swirl it and place it back into the microwave. Heat the agarose/buffer until it
returns to a boil. Remove and swirl. Repeat this process until all of the agarose
has dissolved. When the agarose is heated it will turn translucent but may still not
be in solution. Hold the flask up and carefully look at the solution as you swirl
the flask to confirm that the agarose has dissolved completely.
 Add 8 l of SYBER dye to the gel. Gently swirl and pour the gel solution into the
gel-forming tray. Use a yellow tip to pop any air bubbles found within the gel.
Rinse out the flask and set it next to the sink.

7. When the gel is ready to load, retrieve your tubes (3 of each construct) from the
water bath. To each tube add 5 l of loading dye (to the bottom of the tube) and
pipet gently two times to mix.
8. There are 14 wells in the agarose gel. Load 10 l of Standard #1 in lane #2 and 10
l of Standard #2 in lane #13.

Load 15 l of your 6 samples in lanes #3-#12 - location is your choice - just make
sure that you record it somewhere. You will also be loading 10 l of control vector
(GFP or HA with no insert) cut with the same enzymes. The control should be
grouped with the samples containing the same vector.

9. Run the gel at 120 volts followed by taking a picture of your gel.

During this time, use the vector maps to determine what banding pattern you expect to
see from your gel. Are there restriction enzyme sites within your GOI?

DNA Concentration

Determine the DNA concentration of each sample using the Nanodrop

spectrophotometer. Clearly label the tubes with the concentration.

DNA Sequencing

You will be sending off two of your samples (for each construct) for sequencing. There
will be a signup sheet of the TA bench so that you can label (with black marker only)
your sequencing samples. Label two new clean microcentrifuges tubes on the lid of the
tube and the side of the tube using only the name of the tube (see signup sheet) and
nothing else. Make a dilution of your plasmid DNA so that it is 20 ng/l - you will need
only 20 l of this diluted amount, but can make more if it helps with pipetting small
volumes.

The "stock" tube of your plasmid should be returned to the TAs for storage. It will be
used in a transfection during next week's lab.
 Group 1
• GOI with N-terminal GFP tag
• pAcGFP1-C1 originally cut with HindIII
• Screen with EcoRI and SacI
• GOI with N-terminal HA tag
• pKH3 originally cut with BamHI
• Screen with EcoRI and HindIII

 Group 2
• GOI with C-terminal GFP tag
• pAcGFP1-N1 originally cut with BamHI
• Screen with EcoRI and BstEII
• GOI with C-terminal HA tag
• pKH3 originally cut with HindIII
• Screen with EcoRI and SacII

 Group 3
• GOI (Fragment 1) with N-terminal GFP tag
• pAcGFP1-C1 originally cut with HindIII
• Screen with EcoRI and SacI
• GOI (Fragment 1) with N-terminal HA tag
• pKH3 originally cut with BamHI
• Screen with EcoRI and HindIII

 Group 4
• GOI (Fragment 1) with C-terminal GFP tag
• pAcGFP1-N1 originally cut with BamHI
• Screen with EcoRI and BstEII
• GOI (Fragment 1) with C-terminal HA tag
• pKH3 originally cut with HindIII
• Screen with EcoRI and SacII

 Group 5
• GOI (Fragment 2) with N-terminal GFP tag
• pAcGFP1-C1 originally cut with HindIII
• Screen with EcoRI and SacI
• GOI (Fragment 2) with N-terminal HA tag
• pKH3 originally cut with BamHI
• Screen with EcoRI and HindIII

 Group 6
• GOI (Fragment 2) with C-terminal GFP tag
• pAcGFP1-N1 originally cut with BamHI
• Screen with EcoRI and BstEII
• GOI (Fragment 2) with C-terminal HA tag
• pKH3 originally cut with HindIII
• Screen with EcoRI and SacII