Sequencing at MTU (Taught by Surendar Dhadi)
PCR setup:
x uL water
4 uL sequencing buffer
0.5 uL sequencing primer
100 ng plasmid (if total is 4 kB) or 200 ng plasmid (if total is 8 kb)
0.5 uLBig Dye
-------20 uL
Note: Plasmid must be very pure:
260/230 must be above 1.5
260/280 must be above 1.8
o
o
2 min 95 C, 99 cycles of (26 sec 95 C, 35 sec 46, 3 min 60 oC), 10oC
Store at -20oC for 6 months if desired
Sequencing reaction purification:
- Reuse specific sequencing columns stored in 1M HCl at least o/n to destroy DNA,
spin out HCl, wash 2 x with 500 uL water, ready to load with Sephadex G-50
beads
5M HCl: 15 mL concentrated + 85 mL water
1M HCL: Dilute 5 M 1:5
- While stirring Sephadex suspension with a magnet, add Sephadex 50 suspension to
the columns, let stand a few seconds to let beads sink, remove water from top
(put it back into Sephadex bottle, add more suspension until the columns are
almost full with beads
- Spin the columns 3 min at 0.9 g
- Add one more time Sephadex 50 suspension
- Spin 3 times 3 min at 0.9 g to remove all the water (Orient the columns in the same
way into the centrifuge to not destroy the slant of the Sephadex structure)
- Load the PCR sequencing reaction in the middle of the Sephadex surface
(usually 18 uL are left)
- Place the column into a fresh 1.5 mL tube
- Spin 3 min at 0.9 g (orientation of the column should be followed)
- Now about 13 uL very pure PCR reaction are left
- Add 7 uL water to regain a volume of 20 uL
Loading the Sequencer:
- Use 0.5 mL tubes, put 20 uL clean sequencing reaction in them, and cut the lids off
- Up to 8 reactions can be ran without rubber lids
- If more reactions shall be run, rubber lids have to be put onto the tubes to
prevent evaporation
- Open both doors of the machine
- Check if all buffers and other liquids are filled enough to sequence your amount of
samples, check with someone in Rama’s lab)
- A capillary lasts for 300 runs
- Use only Pop4 polymer, never Pop6
- 500 uL Pop4 are loaded in glass syringe, gives 50 – 60 sequences
- Strictly avoid air bubbles in the capillary
- 2 buffer reservoirs exist, buffer load good for 30 – 40 sequences or 2 – 3 days,
then change buffer
- Push TRAY button -> Tray moves
- Add sequence reactions in a row to the tray, starting A1, the A3, then A5, …
- Push TRAY button again, reactions go inside
- Close both doors
Program the sequencer using the computer:
- Open “310 Data Collection” software
- File>New>Create New, choose “Regular Sequencing” “Sequence Sample Sheet 48
Tubes”
- Enter sample name (Start with A1, then A3, then A5, …)
- Choose “Matrix file”: <none>
- File>Save: Give file name with date
- File>New, choose Sequence Injection List (double-click), Sample Sheet (retrieve the
file
you just saved from a huge list)
- Pull down proper module: P4StDSeq(1ml)E.md4
- Fill down module for all sequences: Mark all (click on the header), then Ctrl-D
- Run time (min) leave at 32 if ,500 bp or set to 40-45 if 1 kb or more
- Injection seconds: Go up to 80 (more sample will loaded, it’s good because we use so
little BigDye)
- Window>Status
- Click the Run button, note the date and time (you will need it to find your sequences
later
- It takes 1 h per sample
Retrieve the data on the computer:
- Start “Sequencing Analysis 5.2” software
- User name = geneseq
- Password = geneseq
- File>Add sample: Choose your folder according to the date and time when your
samples were ran
- Press OK, and my samples will be displayed
- (Select all, click “Clear” button to make the window clear)
- Select: Dye Set Primer: KB_310_POP4_BDTV3_36std.mob (from pull down menu)
- Select: Matrix File: 310Matrix5.mtx (from pull down menu)
- Make selections for all samples (use the Ctrl-D trick as mentioned above)
- Check all boxes in front, and then hit the green Run button
- Colors indicate quality: Blue = excellent, green = good, yellow = bad
- Go to Sequence box, mark all, copy, paste in WordPad
(There must be a way to export the whole data set as ab1 files or something like that)