Editing Phage DNA Isolation (section)

== Qiagen Column Purification<br>  ==

=== Buffers<br>  ===

Qiagen Lambda DNA purification kit (or equivalent)<br>'''Buffer L1''':&nbsp; 20 mg/ml RNAse A, 6 mg/ml DNAse I, 0.2 mg/ml BSA, 10 mM EDTA, 100 mM Tris-HCl, 300 mM NaCl, pH 7.5 (on ice)<br>'''Buffer L2''': 30% polyethylene glycol (PEG6000), 3 M NaCl (on ice)<br>'''Buffer L3''': 100 mM Tris-HCl, 100 mM NaCl, 25 mM EDTA, pH 7.5<br>'''Buffer L4''': 4% SDS<br>'''Buffer L5''': 2.55 M potassium acetate, pH 4.8<br>'''Buffer QBT''': 750 mM NaCl, 50 mM MOPS, 15% ethanol, pH 7.0, 0.15% Triton X-100<br>'''Buffer QC''': 1.0 M NaCl, 50 mM MOPS, 15% ethanol, pH 7.0<br>'''Buffer QF''': 1.25 M NaCl, 50 mM Tris, 15% ethanol, pH 8.5<br> 

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=== Procedure<br>  ===

'''Day 1'''<br>1) Prepare a 150 ml overnight phage lysate culture by mixing 0.5 ml of an overnight culture of E.coli B cells and 10<sup>6</sup> plaque purified phage. This is probably about 10 μl. Add this mixture to 150 ml of media. Grow it overnight at 37 C.<br><br>Purification<br>'''Day 2'''<br>2) Using a glass pipette, add 140 ul of chloroform to the culture to kill the E. coli. Mix gently.&nbsp; Centrifuge the overnight culture at 3900 rpm (Beckman CPR centrifuge) or 5000 rpm (Beckman J2-HC centrifuge) for 5 minutes to pellet unlysed cells and debris.<br> 

<br>3) Save the supernatant by pouring it into a flask. Discard the pellets.<br><br> 

4) Add 216 μl of buffer L1 to the supernatant from step 3. Incubate at 37 C for 30 minutes. After the incubation, save 0.5 ml for later analysis.<br><br> 

5) Add 30 ml of ice cold buffer L2, mix gently, and incubate the sample on ice for 60 minutes.<br><br> 

6) Centrifuge at 6000 rpm for 30 minutes. Discard the supernatant by pouring it out. Drain the bottle/tubes by placing it/them upside down for 1 minute. Do not wipe the inside of the bottle/tubes.<br> 

<br>7) Use a pipette to resuspend the pellet(s) in a total of 9 ml of buffer L3 (not 9 ml per tube).<br>Note that the pellets are probably difficult to see. Pool the solutions into one tube.<br><br> 

8) Add a total of 9 ml of buffer L4. Mix gently (the phages may break easily).<br>Note: If we are short of time, store the sample at 4 C (refrigerator).<br><br> 

'''Day 3'''<br>9) Incubate the tubes at 70 C for 20 minutes, and then let them cool on ice.<br> 

<br>10) Add a total of 9 ml of buffer L5. Mix gently but immediately by inverting the tube 4 to 6 times to mix it well. The solution will become cloudy and viscous. Centrifuge it at 11000 rpm at 4 C for 30 minutes. While the sample is centrifuging, start step 12. When the spin is finished, immediately remove and save the supernatant. Discard the pellet.<br> 

<br>11) If the solution is cloudy, centrifuge the supernatant again at 11000 rpm at 4 C for 10 minutes. Save the supernatant and discard the pellet.<br>Note: If we are short of time, store the sample at 4 C (refrigerator).<br> 

<br>12) Equilibrate the Qiagen-tip 100 with 5 ml of buffer QBT.<br> 

<br>13) Add the supernatant from step 11 to the column. Discard the flow-through.<br> 

<br>14) Wash the column with 10 ml of buffer QC. Discard the flow-through.<br> 

<br>15) Elute the DNA with 5 ml of buffer QF. Save the flow-through which contains your DNA or RNA.<br>Note: If time is short, the procedure can be stopped at any of the steps 15 through 19.<br> 

<br>16) Precipitate the DNA by adding 0.7 volumes of isopropanol. This means that you measure the volume of your sample, multiply it by 0.7, and add that amount of isopropanol. Remember to also precipitate the aliquot saved on step 3.<br> 

<br>Note: If you stop here, put the sample in the refrigerator (4 ̊C).<br><br> 

'''Day 4'''<br>17) Centrifuge for 30 minutes at 10000 rpm. Discard the supernatant.<br><br> 

18) Wash the pellet by adding 5 ml of 70% ethanol for the main sample, or 0.5 ml for the aliquot.<br>Centrifuge at 10000 rpm for 10 minutes. Discard the supernatant again.<br><br> 

19) Add 5 ml 95% ethanol to the sample or 0.5 ml to the aliquot. Centrifuge at 10000 rpm for 10 minutes. Discard the supernatant, invert the tube, and let the pellet air dry for 5 or more minutes. When dry, you will not be able to smell the ethanol.<br> 

<br>20) Add 100 l EB to the sample or 10 l EB (or AE) to the aliquot.<br><br> 

21) For the main sample, take 1 l of the nucleic acid from the previous step and read the A 260 and A 280 in the Nanodrop spectrophotometer. The spectrophotometer will calculate a concentration, assuming it is dsDNA.<br> 

<br>The general rule is that if the phage contains DNA, an absorbance at 260 nm of 1.0 equals a concentration of 50 g/ml. Back calculate how much DNA is in your sample.<br>