Background:
The TOPO TA Cloning System is a commercially available kit that uses a Taq polymerase to generate PCR fragments with a single deoxyadenosine (A) overhang at the
3’ end. The kit comes with a linearized vector that contains a single thymidine overhang at each 3’ end. No DNA ligase is necessary to join fragments. The linear vector comes with a topoisomerase molecule attached. Topoisomerase is an enzyme that cleaves the phosphodiester backbone in one DNA strand. The energy from the broken bond is conserved by formation of a covalent bond between the 3’ phosphate of DNA and the topoisomerase enzyme. This bond between enzyme and the 3’ phosphate can be attacked by the 5’ hydroxyl of another DNA strand, reversing the reaction, and releasing the topoisomerase. This results in covalent attachment of insert and vector DNA without the need for DNA ligase.
In the previous 2 labs the TOPO TA cloning kit was used to introduce PCR fragments into a T vector. In the next 2 labs, the success of the technique will be evaluated by performing several minipreps and checking the DNA by restriction digest and agarose gel electrophoresis. The procedure for minipreps is similar to that used for DNA maxipreps from lab #3. However, each of the reactions is smaller in scale, and the yield should be approximately 2-10
 g of DNA.
Once the plasmid DNA has been purified from the overnight culture of bacteria, the presence or absence of the cloned PCR product will be checked by performing restriction analysis with Eco R1. This enzyme cleaves on either side of the cloned DNA in the T vector. If cloning was successful, the restriction digest should generate a small PCR insert band (about 300 to 500 bp) and a larger band from the excised vector.
In many instances, it is important to purify the cloned DNA from the gel to perform additional manipulations (subcloning, mutagenesis). This can be accomplished by using a razor blade to cut out the band of interest from the gel. The gel can be viewed under UV illumination to help in locating specific bands. The excised band should be as small as possible. It can be placed directly into a SNAP miniprep column and spun in a microcentrifuge to elute a small amount of pure DNA. This method can also be used prior to cloning PCR fragments. In this instance, it might be used to purify one band from several PCR products before adding it to the ligation reaction.
Objectives:
The objectives of this lab are (1) transform E. coli with your cloned plasmid DNA, (2) grow the bacteria and purify plasmid DNA using a miniprep kit, and (3) analyze your cloned DNA by restriction digest and agarose gel electrophoresis. The overall goal is to determine whether you were successful in cloning a PCR fragment. If you are able to clone a PCR product, you will be asked to gel purify the insert using a SNAP column.

Materials:
TOPO TA cloning kit (Invitrogen Corp.) containing T vector and other components required for cloning including the pCR2.1-TOPO vector, 10X PCR buffer, salt solution, dNTP mix, control template, and control PCR primers, DH5

-T1 Competent cells (1 vial
/ transformation), SOC medium.
SNAP miniprep kit with columns
Samples of cellular DNA
37

C shaking incubator
37

C water bath
Disposable gloves
Microcentrifuge
Miniprep kit for DNA purification
Micropipetters
Sterile eppendorf tubes, yellow tips, and blue tips, eppendorf racks
Sterile deionized water
Glycerol and cryotubes for making stocks of overnight cultures
LB broth and antibiotic for overnight cultures
BamHI enzyme and React 3 buffer
TE buffer
Minigel apparatus and combs
Power packs
1.4% agarose in TAE with ethidium bromide
TAE running buffer
100 base pair ladder
6X gel loading buffer
UV illuminator
Gel photography system
Wire loop and Bunsen burner
Methods:
Bacterial cultures:
1.
Start overnight cultures from your bacterial plates. Using a wire loop, carefully remove bacteria from 2 different white colonies. Do this for dishes containing the cloning reaction positive control and for dishes with bacteria transformed by your
PCR products.
2.
Inoculate the bacteria into a 15 ml conical centrifuge tube containing 5 ml of LB medium and 50
 g/ml kanamycin or ampicillin.
3.
Shake the cultures overnight at 37ºC.
4.
Remove 1 ml of bacterial suspension and make a glycerol stock of your transformed bacteria.

5.
Harvest the remaining bacteria by centrifuging the culture in a plastic round bottom centrifuge tube at 5000xg for 5 min. Use the JA-20 fixed angle rotor in the
Beckman centrifuge. There should be a pellet on the bottom side of the tube.
Allow the tube to dry inverted for 5 min and blot excess culture medium.
Miniprep protocol:
6.
Add 400
 l of cell suspension buffer containing RNAse A to the pellet and
resuspend the pellet until the solution is homogeneous.
7.
Add 400
 l of cell lysis solution and mix by inverting the tube several times. Do not vortex. Incubate at room temperature for 5 min.
8.
Add 400 ml of neutralization buffer and mix immediately by inverting the tube 5 times. Do not vortex. You should avoid shearing the high molecular weight DNA.
9.
Centrifuge the mixture at 6000 rpm in a JA20 rotor at room temperature for 10 min. Do not centrifuge at 4ºC.
10.
Pipet the supernate from step 8 into the equilibrated column. Allow the solution in the column to drain by gravity flow, and then discard the flow through.
11.
Wash the columns 2 times with 2.5 ml of wash buffer. Allow the solution in the column to drain by gravity flow after each wash. Discard the flow through.
12.
Elute the plasmid DNA by adding 900
 l of elution buffer. Allow the solution in the column to drain by gravity flow. Do not force out the remaining solution.
13.
Precipitate your plasmid DNA by adding 630
 l of isopropyl alcohol to an eppendorf tube. Centrifuge the mixture at 12,000 xg at 4ºC for 30 min. Use an eppendorf centrifuge at top speed.
14.
After the ethanol has evaporated, (15 min) dissolve the DNA in 100
 l of TE buffer. Allow at least 10 min to dissolve completely. Occasionally, insoluable particles are present, but these do not influence the quality of DNA.
Determination of DNA concentration:
15.
Measure the absorbance of the DNA at 260 nm to determine the concentration.
Turn on the spectrophotometer and press dsDNA.
11. Fill the quartz cuvette with 700
 l of water and blank the instrument.
12.
Add 700
 l of TE buffer to an eppendorf tube, add 7
 l of sample DNA from the miniprep and mix thoroughly.

13.
Add the sample to the quartz cuvette, carefully place the cuvette in the spectrophotometer, and close the cover. Touch the sample button and allow the instrument to determine absorbance. Record the value.
14.
Calculate DNA concentration by multiplying absorbance (should be about 0.04 to
0.08) by 5000 (100 sample dilution x 50 conversion factor). This will yield the
 g/ml of DNA. 20
 g is a good yield.
Restriction digest:
15.
Set up 4 eppendorf tubes on ice for restriction digest as shown in the table below.
Add about 1
 g of each of your plasmid DNAs to tubes 1-4.
DNA sample control-1 control-2 sample-1 sample-2 amt DNA
1-17
 l
“
“
“ amt React 3
2
 l
“
“
“ amt water up to 19
 l
“
“
“ amt enzyme
1
 l
“
“
“
16.
Add 2
 l of React 3 buffer to each of the tubes.
17.
Add deionized water to each tube to bring the total volume to 19
 l. Mix contents.
18.
Add 10 units (1
 l) of Eco RI to each tube. Mix enzyme well. Briefly spin tubes to pellet reaction and incubate tubes at 37ºC for 1 hour.
19.
As soon as the reaction is incubating, start to pour a minigel. This should contain
1.4% agarose in 1x TAE buffer with ethidium bromide. Pour the gel such that it is
3-4 mm thick. Hook up the gel to the power pack to make sure that all leads are working. As soon as the restriction digest is finished, add 4
 l of 6x gel loading buffer to each tube and load the gel. Run the gel at 90 V. The vector is about
4000 bp and the control insert is 500 bp.
Purification of cloned insert using a SNAP column:
20.
Place your agarose gel on a UV light box and cut out the small insert band using a sharp razor blade. Try to cut out as little agarose as possible.
21.
Place the slice of agarose in a SNAP column and centrifuge at full speed for 10 seconds. The material eluted from the column contains purified DNA.
22.
Use one half of the eluted DNA in an agarose gel to ascertain that the band has been purified. This purified DNA could be used directly in a cloning reaction.