 Circularize DNA oligos of very small size - 

The g-complex loads the b-clamp to the p/t and b-clamp then slides on the DNA bidirectionally. In order to ensure that the b-clamp does not slide off the edge of the p/t one can either block the ends of the DNA using biotin-streptavidin or use circular DNA. In our unpublished experiments, we see significant inhibition of polymerase activity due to the presence of bulky groups like streptavidin, hence we proceeded to use circular template for our work. 

Observing in-vitro DNA synthesis using beta clamp

DNA oligonucleotides were purchased from Integrated DNA Technologies (Coralville, IA, USA) and their sequences. All oligomers were purified in-house using denaturing PAGE purification. The templates contained a 5’ phosphate modification in order to circularize the template. Briefly, the template to be circularized was annealed to a 20 mer linker, which was complementary to the first ten and the last ten nucleotides of the linear template. After annealing, we used T4 DNA ligase on the template/linker to connect the 5’-P and 3’-OH, forming a circle. The circle was then purified of its linear product and the linker by running through a PAGE gel and subsequent extraction.

- circular template - 5' Phosphate modification
- linker DNA 2X Circular template :**  **complementary to the first ten and the last ten nucleotides of the linear template.
- Anneal
- Add T4 ligase 1 microL for 1 microgram
- 20Hours 20C
- ethanol precipitation
- **purify on 10% PAGE gel - poured into a SDS protein gel**
-  

small DNA/oligos Circularization and purification

Mass spectrometry is a commonly identification method for the products of a protein digest, modification, purification, or the like. Sample preparation is handled via Ziptips, an extremely fast (if not expensive) method of reducing contaminants in your sample.

ZipTipping solutions

A. 	0.2% TFA in HPLC grade water; for dilution of protein digests.

B.	0.1% TFA in 50% ACN/50% water; for peptide elution.

C. 	100% acetonitrile (ACN); wetting agent for ZipTip.

D. 	0.1% TFA in water; for equilibration of ZipTip

 

1. For each unknown, label a 0.65 ml tube as a diluted sample. Combine 6.0 µl of solution A and 4.0 µl of your protein sample. Gently mix, and then cap the tube. 

2. Transfer 2.5 µl of solution B to the bottom of the tubes and cap them. Label as elution tubes

3. Attach a ZipTip to your pipettor and set volume to 8 µl.  Draw up 8 µl of solution C and dispense onto a Kimwipe.  Keep the plunger down, and repeat once.  Proceed to step #4 without letting the plunger retract, i.e. without sucking air through the C18 material.

4. With the plunger still down, draw up 10 µL of solution D and dispense onto a Kimwipe. Keep the plunger down, and repeat 2X.  Proceed to step #5 without letting the plunger retract.

5. Slowly and carefully cycle 8 µl of your diluted sample from #1 above through the equilibrated ZipTip.  Do 10 complete cycles while keeping the tip immersed to bind the proteins to the tip. At the end of your last cycle, dispense the liquid into a Kimwipe. 

6. Place the tip in solution D and draw up 8 µl.  Dispense onto a Kimwipe and repeat 3X.  This step is critical for removing non-peptide components. After the last wash, make sure all the liquid is out of the tip.

7. Set the pipette for 2.0 µl and elute the peptides by cycling the solution B in the PET tube (from step 2) through the tip for a total of 6 cycles.  Cap the tube tightly and store at -15 to -20°C.

MALDI-TOF Protein Prep

A common protocol for the purification of polyhistidine tagged proteins uses an Immobilized metal affinity column (IMAC). This protocol for affinity purification is a generalization that utilizes Amersham sepharose beads and nickel ions.

Buffers
Binding Buffer -  pH 8.0 with monosodium phosphate and NaOH, and 20mM Imidazole to reduce nonspecific binding. 

Wash Buffer - pH 8.0 with monosodium phosphate and NaOH, and 60mM Imidazole to reduce nonspecific binding.  

Elution Buffer - pH 8.0 with monosodium phosphate and NaOH, and 400mM Imidazole to reduce nonspecific binding. You can optionally use a pH 6.0 buffer that elutes the metal ions from the agarose beads, but this introduces metal ion contamination to the elution.

Column Preparation
1. In 4 C cold room, set up the column on a vertical support. 

2. Flush the column with cold ddH2O. Leave some water in the column. 

3. Resuspend the Amersham sepharose. Transfer the sepharose to the column using a pipette with the tip cut off. 

4. Open the outlet for the column to allow the sepharose to pack at the bottom. Do not let the sepharose dry out.

5. Wash the column with 5 volumes of ddH2O. Drain the last wash to just above the sepharose.

6. Add two column volumes of 0.1M NiSO4. Drain the NiSO4.

7. Wash the column with 5 column volumes of ddH2O until the sepharose is blue-green.

8. Wash the column with 5 column volumes of binding buffer until the sepharose is blue. 

Lysate Preparation

1. Thaw cell pellet from 250 ml culture.

2. Resuspend pellet in 15 ml Binding buffer and 1.0 mM PMSF (150 ul of 100mM).

    a. Binding buffer is pH 8.0 with monosodium phosphate and NaOH, and 20mM Imidazole to reduce nonspecific binding. 

4. Add the resuspension to prechilled oakridge tubes. 

5. Sonicate the cell suspension 10 seconds at a time, with 10 seconds in between sonications. Repeat 7 times.

6. Centrifuge the lysate in SS34 rotor at 18,000 rpm (39,000 RPM) for 20 minutes at 4 oC.

7. Filter the lysate supernatant through a 0.22 um syringe filter.

Immobilized Metal Ion Affinity Chromatography

1. Apply lysate to the prepared column. Collect the flow through in a tube and reapply to the column.

2. Repeat step 1 two more times. 

     a. Optionally, you can save 100 ul of the flow through for analyzing initial binding conditions. If so, add 2X laemmli buffer to the 100 ul and freeze at -20 oC.

3. Prepare 50 ml (10 column volumes) of wash buffer with 0.1 mM PMSF (500 ul).

4. Add the wash buffer to the column, draining 5 ml fractions.

     a. Optionally save these fractions by freezing them at -80 oC.

5. For fractions 2, 5, and 8, transfer 10 ul of the fraction to a microfuge tube and add 2X Laemmli buffer. Freeze these tubes at - 20 oC.

6. Prepare 10 ml elution buffer (10 column volumes) with 0.1mM PMSF (250 ul).

7. Add 5 ml elution buffer to the column and allow it to incubate for 5 minutes before allowing it to wash through. 

8. Collect the elution in 1 ml fractions using 13x100 mm disposable glass tubes. Save all fractions by freezing them at -80 oC.

     a. Optionally save 10 ul of each elution fraction by adding 10 ul 2X Laemmli buffer in a microfuge tube. Freeze these tubes at -20 oC.

Wash the column with 10 volumes of binding buffer. If storing the column for a long period of time, wash the column with ddH2O and store the sepharose in 20% ethanol.

IMAC Protein Purification

Here is a scraping protocol for large scale induction and harvesting of transformed E. coli (typically BL21 or NiCo21 strains), starting with already transformed colonies. For expression of some proteins, scraping bacterial lawns from plates seems to increase the overall yield. Frozen cell pellets can be stored almost indefinitely in a -80 freezer until they are needed for lysis and protein purification. 

1. Inoculate 3 transformed colonies in 200 ul LB-amp-cam broth. 

2. Plate the 200 ul broth mixture on LB-amp-cam plates. Incubate overnight at 37 oC. 

3. You can prepare additional plates in this step if desired to decrease the time needed to reach the necessary optical density

4. Prewarm 1 L LB amp/cam in a 2 L fernbach flask at 37 oC. 

5. Scrape culture from plate media to the broth until an OD600 of 0.1 is reached. .

6. Grow the cells at 37 oC on a 225 RPM shaker to an OD600 of 0.5-0.8. Check the OD600 frequently using 1 ml of culture in a cuvette to prevent overgrowth.

7. Add 10 ml 100 mM IPTG to the 1 L culture and grow for 2 hours.

8. Spin cells down 200 ml at a time in 250 ml sorvall bottles using the Sorvall RC5 centrifuge. Spin for 5 minutes at 6500 G and 4 oC.  Perform multiple spins to spin down all culture media.

9. Freeze cell pellets at -80 C.

IPTG Induction for Protein Expression

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