Sequencing on the BaseStation

 

For Plasmids: Use 500 ng DNA in 8.3 uL ddH2O

 

1/8 BIG DYE Reaction PCR Conditions
PCR Reagents amount/reaction final concentration
DNA + ddH2O 8.3 uL 500 ng 96°C 30 seconds
5X dilution buffer 0.6 uL see below 96°C 10 seconds
Primer 0.1 uL 2 pmoles 55°C 15 seconds

30 X
Big Dye Mix 1.0 uL 1/8 60°C 4 minutes
10 uL 4°C ¥

 

5 X dilution Buffer-
400mM Tris HCl pH 9.0
10mM MgCl2

-Add 0.632 g Trizma-HCl to 10 mL ddH2O
-Adjust pH to 9.0
-Add 0.02 g MgCl2
Can be stored at Room Temp or 4 degrees.

 

 

Cleanup:

Prolinx RapXtract plates:

Plates can be stored at room temp for short term or 4 degrees for long term

1. allow plate to warm up to room temperature (at least 15 minutes)

2. place the plate on the super high powered magnetic plate

3. cut and carefully pull away the plastic film from only the number of wells you need

4. while it is still on the magnet, invert the plate onto a paper towel to get rid of the storage buffer from the wells you will use

5. add your samples to the wells

6. remove plate from magnet and place on vortex with special 96-well plate adapter

7. vortex at the designated setting (indicated with tape; vortex setting 3.5) for 20 seconds

8. place back on the magnet and suck out clear product (a little bit of the beads is ok).  Store plate with film on unused wells for later use

9. add 5-10 uL (this amount can vary) of 1X crystal violet loading dye, denature at 65°C for 3 min, quench on ice and load onto BaseStation.

 

 

 

Ethanol Precipitation:

1.      For a 10ul Reaction add 30ul of EtOH/NaOAc (*see below*) mix to each well. 

2.      Centrifuge at 4 C for 30 minutes at 3200 rpm.

3.      Decant and allow sample plate to dry upside down on a clean paper towel for 3 to 5 minutes.  It is important to keep the plate upside down after decanting so as not to allow the EtOH to   get back into the sample.

4.      Turn the plate back over and add 50ul of 70% EtOH.

5.      Centrifuge at 4 C for 10 minutes at 3200 rpm.

6.      Decant and allow sample plate to dry upside down on a clean paper towel for 3 to 5 minutes.

7.      Place slanted upside-down in fume hood to dry (about 2 hours) or turn upside-down on a paper towel in centrifuge and spin at 500 X g for 1 minute.

8.   Resuspend in 5uL 1X crystal violet denature at 65°C for 3 min, quench on ice and load onto BaseStation.

 

*To make 3M NaOAc dissolve 408.24g of NaOAc – 3H2O in 800 ml of ddH2O.  Spin until everything is into solution.  Then titrate mixture to a pH of 4.8 using glacial acetic acid.

*To make  EtOH/NaOAc premix for 100 ml total add 94ml of 95%EtOH and 6ml of 3N NaOAc.