This is a protocol Allan DeBono used when cleaning BigDye 3.1 sequencing reactions for sequencing at NAPS. In both of our hands it worked consistently to confirm vector inserts. An advantage for this protocol is that it takes 5-10 minutes to preform depending on how many reactions are being run (and how fast of a pipetter you are). It also utilizes spin columns that can be re-used after autoclaving.
The reaction costs about ~$0.20 each for a sephadex column (non-academic pricing) and ~$0.89 including a fresh basket-column (re-using columns is possible). Prices are subject to change with suppliers and availability of the mentioned products below. This saves a decent amount in the long run when ordering user-prepared/user-cleaned sequencing reactions ($2/3 each) compared to user-prepared NAPS cleaned ($4/5 each) reactions or sequence drop-off ($10/7). There is an additional cost if you buy individual BigDye 3.1 ready mix ($1.55 each) but as of right now thanks to Marco’s efforts, we have a surplus of reactions and dilution buffer in our freezer. If you are cleaning up to 24 samples the cost savings increase dramatically.
For 24 samples its around $4.80 for clean up (re-using baskets), plus $48 for sequencing = $52.80 for User-Prep & User-Clean vs. $96 for User-Prep/Nap-Clean or $168 (3.2x the cost) for Naps-Prepared. For more reactions at a time consider using a magnetic bead clean up as it may be less expensive and easier to handle for larger sample qualities.
Materials
- Sephadex G-50 DNA grade Fine (GE Illustra Product code 17-0573-02)
- 500 mL 10 mM Tris-EDTA DEPC-treated
- Big Dye Reaction
- DEPC treated (autoclaved) distilled water
- Autoclaved Filter basket mini-columns (USA Scientific Product ID: 1415-0600)
- Sterile 1.5 or 2 mL tubes
- Sterile single or 8-strip PCR tubes
Protocol
Prepare Sephadex-50 6%
Weigh 6 g (swells about 9-11 mL per gram) into a 30 mL of 10mM Tris-EDTA DEPC-treated.
Mix well and let settle out/rehydrate partially (30-45 minutes)
Add more 10mM Tris-EDTA DEPC-treated to a volume of 80 mL
Mix well and let settle out/rehydrate fully (30-45 minutes)
Remove layer of cloudy solution down to resin bed
Refill with 10 mM Tris-EDTA DEPC-treated to 100mL and repeat 1-2 times until solution stands clear
Make a final solution of 6% 10 mM Tris-EDTA DEPC-treated (6g/100mL)
Store in 4℃ for up to 4 weeks or -20℃ up to 6 months
***This makes up ~115 6% sephadex columns so consider making up a 1/2 batch if it will not be used within 6 months as sephadex G-50 is expensive to discard in bulk.
Packing 6% Sephadex columns
Mix 6% solution well prior to use
Cut a P-1000 tip about 1/3 up the conical tip (This helps with a quick and even draw up of solution, otherwise its a long pipetting process due to a high viscosity of the solution)
Load 600 µL of 6% solution into a filter basket mini-column
Spin down at 8000g (RCF) for 1 minute
Hydrate with 600 µL ddH20 (or preferably DEPC)
Spin down at 8000g RCF for 1 minute
Place column into a fresh 1.5 or 2 mL tube
Mix 10 µL of sequencing reaction with 10 µL of ddH20 (or preferably DEPC)
Load 20 µL into the centre of the filter plug
***Do not let the reaction run down the inside of the tube or disrupt the filter matrix with the pipette tip. It helps to hold the column at a 45 degree angle and pipette down onto the flat surface of the plug (vertically the column has a slant from being centrifuged)
Spin down at 8000g RCF for 1 minute
Collect eluted volumes into an 8-strip PCR tube
Adjust final volume to 15-20 µL (depending on quantity of initial DNA sample)
Check quality and relative concentrations with nano-drop (optional)
Turn in 20 µL of labeled sequencing reaction to NAPS
Pop out the Sephadex G-50 plug for discarding and soak the filter baskets in DEPC water over night. Air dry at room temp and autoclave prior to use.
Happy sequencing!
-Dylan