Background

The goal of this laboratory is to purify plasmid DNA from E. coli. A plasmid is a small double-stranded, circular DNA molecule that replicates independently of chromosomal DNA. A plasmid must contain an origin of replication, and may contain promoters, antibiotic resistance genes, other coding sequences and poly-linker regions. Plasmids are used for cloning vectors and for protein overexpression. The plasmid you are purifying here is pYP, derived from pDEFEND2, which is in turn derived from pUC19. Each of these plasmids contains a gene that confers resistance to the antibiotic ampicillin. pDEFEND2 also contains a promoter for T7 RNA Polymerase followed by a polylinker. pYP contains an insert into the polylinker that codes for yeast-Phe-tRNA (pYP actually contains a mutant of yeast-Phe-tRNA). In a later lab you will use a fragment of your plasmid as a template to synthesize the RNA.

There are many different protocols for isolating plasmid DNA from bacterial cultures. One common and reliable procedure, which is used here, is known as the alkaline lysis method. In the alkaline lysis method, bacteria are grown in LB broth and are pelleted by centrifugation. The pellet is suspended in an alkaline solution (NaOH) to lyse the bacteria and to denature proteins and DNA. The solution is neutralized in potassium acetate. Bacterial proteins aggregate and precipitate, entangling large chromosomal DNA fragments, which are too large to renature. The plasmid DNA is relatively small, and does renature and remain free in solution. Following centrifugation, the plasmid DNA (in the supernate) is further purified chromatographically.

You will receive a pellet of E. coli from your TA. After purification of the plasmid, you will linearize the plasmid with restriction endonucleases to confirm the identity of the plasmid. Restriction endonucleases recognize and cut specific DNA sequences. In addition you will perform a larger scale, preparative restriction digest. That restriction reaction takes 16 hours, so you will let it run overnight. The product of that reaction will be used in a following experiment as a template for synthesis of RNA with T7 RNA polymerase.

Supplementary Reading
Voet & Voet: Nucleic Acid Manipulation 28-3.A, 28-3.C, 28-4.C, 28-5.A, 28-5.B, 28-6.A & 28-8.A.

Qiagen Handbook : Read midi and maxi prep protocol, p.12-15 and "General Considerations for Optimal Results" p 56-66 in detail.

Prelab
Write out a brief synopsis of the procedure in point form.

Materials for plasmid isolation
You will be using a QIAGEN Maxi-Prep column kit to purify your protein. The instructions come from the handbook included with the kit.  You will use buffers from the Qiagen kit (Warning: One of the buffers contains the enzyme RNAse that chews up RNA. Care should be exercised when using this buffer to avoid contaminating the lab with RNAse. RNAse contamination will make your transcription reaction difficult.
You must wear gloves throughout this lab to protect your DNA/RNA.

Methods for plasmid isolation

(performed by TA)

1) Isolate a single colony from a freshly streaked selective plate and inoculate a 5 mL LB/AMP culture. Incubate for ~8 hours at 37 deg Celsius.

2) Dilute the culture 1/100 into LB medium that contains ampicillin by inoculating 100 mL cultures with 0.1 milliliters. Grow at 37 deg Celsius for 12 hours.

3) Pellet by centrifugation at 6000 x g for 15 minutes at 4 deg C. The pellets can be maintained overnight on ice.

(performed by student)

4) Suspend the bacterial pellet in 10 mL of Buffer P1 . Be sure to fully disperse the pellet. Buffer P1 must be cold (on ice) before use.

5) Add 10 mL of Buffer P2 (NaOH/SDS) to the suspended pellet. Close the Buffer P2 bottle immediately to prevent contamination. Before using buffer P2 you may need to warm it 37 deg C to insure that the SDS is in solution. Mix the suspended pellet gently but thoroughly, by inverting 4-6 times. Incubate at room temp for no more than 5 minutes. Your solution may become viscous. Do not vortex. That will shear the genomic DNA.

6) Add 10 mL of chilled Buffer P3 (ammonium acetate). Mix immediately but gently by inverting 4-6 times, and incubate on ice for 20 minutes. Your solution may become cloudy and viscous.

7) Centrifuge at ~20,000G for 30 minutes at 4 degrees C in centrifuge tubes provided by your TA. Your supernatant will be clear after centrifugation. Keep the supernatant (which contains the plasmid DNA) and discard the pellet.

8) After ~20 minutes of centrifugation, equilibrate a QIAGEN-tip 500 by applying 10 mL Buffer QBT. Allow the column to drain by gravity flow. You may let the column run unattended while you wait.

9) Apply the supernatant from step 9 to the QIAGEN-tip and allow it to enter the resin by gravity flow. This setup MUST be performed ASAP after the supernatant has been centrifuged to prevent protein precipitation. During this step the DNA will bind to the column. The DNA will remain bound during washes with low salt QC buffers. It will elute in the high salt buffer QF.

10) Wash the QIAGEN-tip once with 30 mL Buffer QC.  Be sure that the salts have not precipitated out of the QF solution, if they have, warm to 37 deg C for about 10 min.

11) Elute with 15 mL of Buffer QF. Do not use a polycarbonate tube for collection since they are not alcohol resistant.  Use special tubes provided by TA.

12) Precipitate the DNA by adding 10.5 mL isopropanol. Mix and centrifuge at 15,000 x g for 30 minutes at 4 deg C. Gently decant the supernatant (the pellet can be easily dislodged).

13) Wash pellet (you probably can't see it) with 5 mL 70% ethanol and centrifuge at 15,000 x g for 10 minutes.

14) Air dry the DNA and dissolve in 1 mL 10 mM Tris-HCl, pH 8.0. Take 10 microliters, dilute to 200 microliters with Tris-HCl. pH 8.0. Determine the absorbance spectrum from 360 to 220 nm.

15) Determine the yield (micrograms/mL)= OD(260nm) x 20 x 50 micrograms.

16) Save 20 microliters for the gel, which you will run the next week.

Materials

Ear I restriction endonuclease

10X NEBuffer 1 (supplied with enzyme)

DNA that you just purified

Methods

1) To prepare your plasmid for preparative digestion and transcription, label a 1.5 mL Eppendorf tube with your name and "Ear1 digest". Mix 120 microliters DNA (from #15 above), 12 microliters 10x NEBuffer 1, and 1.5 units Ear I for each 1 microgram of DNA.

2) Tap the eppendorf tube gently to mix.

3) Incubate the Ear I eppendorf at 37 C for 16 hours. Your TA will stop the reaction and perform steps 8 & 9.

4) To prepare your plasmid for analytical digestion and electrophoresis, label three eppendorf tubes with your name and: E (Eco RI), H (Hae II), and B (Bss HII).

5) Place 10 microliters of purified DNA solution (from #15 above) in each tube.

6) Give the tubes to your TA who will store at -20 C until next week.

7) Label the remaining DNA sample with your name, and 'yeast-phe unmodified tRNA' and give it to your TA.

(performed by TA)

8) Inactivate Ear I by heating to 65 degrees for 20 minutes.

9) Store at -20 C until next lab.

1) Define the following terms:

a) vector

b) sticky ends

c) restriction endonuclease

d) ligation

e) competent cells

f) polylinker region

g) heat inactivation

2) What is the purpose of the gene for ampicillin resistance in the plasmid?

3) In the plasmid shown above, there is a restriction site for EAR I. Indicate the position of the cut(s).

4) Go to http://www.neb.com. Follow the links to the Restriction Endonuclease catalog. From this, what sequence does EAR I recognize and where in that sequence does it cut?

5) We might have prepared the desired product using the Polymerase Chain Reaction. In about 1-2 paragraphs, explain this process.

6) Draw two of the base-pairing interactions in the anti-codon stem of yeast-phe-tRNA. Indicate the residue numbers. Measure the distances of the hydrogen bonds.