PCR Protocols
Crossover PCR (click here)
PCR cleanup with AMPure magnetic beads
1. For each template make 400 ul of 70% EtOH. This is done by using a fixed volume of 100 or 95% ethanol and a fixed volume of H2O. Because of solvation the final volume will be less but it doesn’t matter. Do not make up the solution by adding an amount of ethanol into a graduated cylinder or tube and then adding H2O to a certain volume. Because of solvation you will be diluting the ethanol beyond 70% and this may effect yields.
2. Resuspend the AMPure beads (4ºC fridge) (not the CleanSeq beads). Do not vortex, instead flick bottle with fingers.
3. The amount of AMPure beads to add is determined by the formula :
1.8 x (reaction volume) = volume of beads per reaction. If there has been a
volume loss then the amount of beads should be adjusted accordingly.
| PCR reaction volume in ul | Amount of beads to add in ul |
| 25 | 45 |
| 50 | 90 |
| 75 | 135 |
| 100 | 180 |
4. Adjust pipettor to 75-80 ul, pipette up and down 10X. The PCR products are stable in the solution and can be left for a short while. Save tips for step 6.
5. Place on magnetic plate for 5-10 minutes, depending on volume of PCR product.
6. With tubes on plate slowly take off supernatant. The pellet may be somewhat soft. Transfer to non-magnetic plate.
7. Add 200 ul of freshly made 70% ethanol, allow to sit for 30 seconds. If using the same set of tips for all washes, triple rinse in 70% ethanol before using in a different row.
8. Place on magnetic plate for 3 minutes.
9. Take off supernatant.
10. Repeat steps 7-9, taking off as much supernatant as possible
11. The biotech center recommends going to step 12. (I allow them to dry by placing them in a 37ºC incubator for ˜15 min.) [This is also a good step to store them sealed, at -20 or 4ºC.]
12. Place on non-magnetic plate. Add 40 ul of EB buffer from the Qiagen kit or 10mM Tris-Cl (pH 7.5-8.0) or Tris-Acetate pH 8.0. (I usually tap the tubes so the liquid goes to the bottom)
13. Place on magnetic plate for 5-10 minutes.
14. Withdraw 38 ul to a new tube.
Gel Band Clean Up - from Mehtap Yildiz, Oct 2007
This protocol is useful for cleaning small fragments, such as those from qRT-PCR, for sequencing. The recipes for the required solutions are given below the protocol.
1. Cut DNA fragments from gel and place in 1.5 ml microfuge tube.
2. Add 400 ul of Ultra Salt to each tube.
3. Incubate tubes in 60C waterbath, shaking often to melt gel fragments.
4. Virtex Ultra Bind (Silica) for 30 sec.
5. Add 10 ul of ultra Bind to each tube and mix by flicking the tube.
6. Incubate at room temp for 5 min.
7. Centrifuge at high speed for 5 sec.
8. Remove supernatant and discard.
9. Resuspend pellet in 1 ml of Ultra wash and vortex for 10 sec.
10. Centrifuge at high speed for 5 sec.
11. Remove supernatant and discard.
12. Centrifuge at high speed for 5 sec and remove residual liquid by dapping it with a kim wipe
13. Resuspend pellet in 20 ul of sterile milli-Q water and mix gently by pipetting.
14. Incubate at room temp for 5 min.
15. Centrifuge at high speed for 1 min.
16. Transfer supernatant, which contains the DNA, to a new tube. This DNA can be used for sequencing with the following reaction:
water 4.25 ul
10X buffer 1.0 ul
Big Dye 0.75 ul
Primer (5 uM) 2 ul
DNA 2 ul
95C for 3 min; 50 cycles of (95C for 20 sec, 50C for 20 sec, 60C for 4 min); 72C for 5 min; 4C hold
Solutions:
6 M NaI (149.89 g/mol) = Ultra salt
To make 500 ml, dissolve 449.67 g of sodium iodide in water and bring to a final volume of 500 ml. Filter through a 0.45 um filter.
50 mM NaCl, 10 mM Tris-HCl pH 7.5, 2.5 mM EDTA, 50% (v/v) ethanol = Ultra wash
To make 1 L, mix
1 ml of 1 M Tris pH 7.5
0.5 ml of 0.5 M EDTA
485 ml water
56.5 ml of 100% ethanol
DNA binding matrix:
Add 12.5 g silica (Sigma S-5631) to a tall cylinder and suspend in 250 ml of water. Let stand overnight. Pour off cloudy supernatant. Add water to 50 ml. Add 50 ul of concentrated HCL and autoclave. Aliquot to sterile 1.5 ml tubres. May be stored at room temperature.