DNA sequencing
Sequencing Cleanup with CleanSeq magnetic beads
1. For each template make 200 ul of 85% ethanol. (Agencourt recommends 85%; the biotech center recommends 80%) 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 in a graduated cylinder or tube and then adding H2O to a certain volume. Because of solvation you will be diluting the ethanol beyond 80% and this may effect yields. For labeled fluorescent fragments use 100% ethanol. Also make up or have on hand 40 ul/reaction sterile H2O or 0.1mM EDTA or 0.5mM EDTA.
2. Resuspend the CleanSeq beads (4ºC fridge) and add 10 ul. Do not vortex, instead flick bottle with fingers.
3. Add 62 ul of freshly prepared 85% ethanol. (The biotech center recommends 80 ul of 80%; I can’t tell the difference.) Adjust pipettor to 75-80 ul.
4. To mix pipette up and down 7X. Save tips for step 6.
5. Place on magnetic plate for 3 min. Solution should be clear.
6. With the tubes on the magnetic plate slowly withdraw the liquid. Transfer to non magnetic plate. Add 100 ul 85% ethanol, allow to sit for 30 seconds. (The biotech center recommends 150 ul of 80%) If using 4 ul or more of Big-Dye a second wash may be necessary.
7. Place on magnetic plate for 3 min; withdraw as much liquid as possible.
8. The biotech center recommends going to step 9. (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.]
9. Add 40 ul of 0.1 mM EDTA, 0.5 mM EDTA or H2O, [The Biotech center recommends 50 ul] (I usually tap the tubes so the liquid goes to the bottom, incubate for 5 minutes. Optional: replace on the magnetic plate for three minutes and transfer supernatant to new tubes). EDTA should be used if H2O elution yields capillary signal values above 400 RFU.
10. For capillary sequencing remove 20 ul to a strip tube labeled with a tough
tag and take to the biotech center.
If using a multi-channel pipettor, the bead manufacturer says that changing
tips between washing steps is probably unnecessary. However triple rinse the
tips in 70% EtOH before using in a different row.
Sequencing Reactions with Big Dye
2 ul Big Dye
4 ul 5X buffer
4 ul plasmid DNA
1 ul primer (10 uM stock)
Step 1: 95C for 3 min.
Step 2: 96C for 15 sec.
Step 3: 58C for 4 min.
50 cycles of steps 2 and 3
Step 4: 72C for 7 min.