Editing PEG Precipiate PCR Product

'''PEG Precipitation of PCR products'''

1, Run 50 ul PCR

2, Add 50 µL of PEG to a 0.5 mL tube. Transfer the remainder of the PCR to the tube with PEG and mix by pipetting up & down very well. 

3. Let the PCR + PEG mixture incubate at 37°C for 15 min.

Place bottle of 80% EtOH on ice to keep ice cold. 

4. Centrifuge PCR + PEG at high speed (~15,000 x g) for 15 min. at room temp.

5. Using a P200 pipetter, pull off the supernatant & discard it.

6. Add 125 µL of cold 80% EtOH to the side ot tube (do not add EtOH directly to the bottom of the &nbsp;tube). 

Let the tube sit for one minute. alternatively, spin at max speed at 1 min.

Using a P200 pipetter, pull off the supernatant & discard it. 

7. Repeat step 5~6

8. Dry off the EtOH by evaporation at 37 oC &nbsp;for 5-10 min. There should be no trace (visible or by smell) of EtOH when done. 

9. Dissolve the PCR product in 25 µL of water or TLE. 

Pipette up & down several times to ensure the DNA has gone into solution. it is also usful to let &nbsp;it sit for several minutes,&nbsp;



=== '''PEG Solution''' ===

10.0 g Polyethylene glycol 8000 (MW = 6000 - 8000 is fine)(20% PEG) 

7.3 g NaCl (2.5 M NaCl) 

ddH2O up to 45 mL - shake & let PEG go into solution. 

Note: PEG takes 20+ min to go into solution when you make the initial solution. after everything is in solution, fill with ddH2O up to 50 mL. 

===  ===

=== '''TLE''' ===

10 mM Tris, 

0.1 mM EDTA 

80% EtOH 



=== '''50X TAE Stock''' ===

Solution For each litre of solution: 

242 g Tris Base (MW=121.1) 

57.1 mL Glacial Acetic Acid 

100 mL 0.5 M EDTA 

mix Tris with stir bar to dissolve in about 600 mL of ddH2O. 

add the EDTA and Acetic Acid. 

bring final volume to 1 L with ddH2O. 

store at room temperature. 

Note: Final (1x) working concentration : 

0.04 M Tris - Acetate 

0.001 M EDTA 



=== 50X Modified TAE Stock Solution  ===

For each litre of solution: 

242 g Tris Base (MW=121.1) 

57.1 mL Glacial Acetic Acid 

10 mL 0.5 M EDTA 

mix Tris with stir bar to dissolve in about 600 mL of ddH2O. 

add the EDTA and Acetic acid, pH to 8.0 with additional Acetic acid bring final volume to 1 L with ddH2O.

store at room temperature. 

Note: Final (1x) working concentration : 0.04 M Tris - Acetate 0.1 mM EDTA (this is 1/10 of the normal TAE EDTA concentration)






[[Category:Protocols]]