Sequencing Rxn 1 of 2

Sequencing Reaction

(following miniprep of plasmid DNA)

A forward and a reverse reaction is to be prepared in the following manner:

Forward Reaction:tube__tube__tube__tube__ etc.

  1. Sterilized SDW***__ul__ul__ul__ul
  2. Big Dye buffer*4 ul4 ul4 ul4 ul
  3. Forward Primer*2 ul2 ul2 ul2 ul
  4. DNA template (after miniprep)__ul__ul__ul__ul
  5. Big Dye Terminator Mix**1 ul1 ul1 ul1 ul
  6. TOTAL VOLUME20ul20ul20ul20ul

* located in -20˚C in box labeled “Universal Primers”

** located in -20˚C/top fridge labeled “PCR sequence” – brown tube labeled “BDT”

*** Add SDW up to 20 ul

Reverse Reaction:tube__tube__tube__tube__ etc.

  1. Sterilized SDW__ul__ul__ul__ul
  2. Big Dye buffer4 ul4 ul4 ul4 ul
  3. Reverse Primer2 ul2 ul2 ul2 ul
  4. DNA template__ul__ul__ul__ul
  5. Big Dye Terminator Mix1 ul1 ul1 ul1 ul
  6. TOTAL VOLUME20ul20ul20ul20ul

Sequencing Reaction Conditions:

1. 96C 2 min

2. 96C 30sec

3. 50C 15sec

4. 60C 4 min

5. Goto Step 2 Repeat 24X

6. Hold at 15C

NOTE:

  1. Big Dye Terminator mix is obtained from Denise (~100ul), and 2ul was aliquoted into small PCR tubes…prepared on ice.
  2. Make a Master mix containing SDW, Big Dye buffer and Forward Primer for Forward reaction and Reverse Primer for Reverse reaction. Make 1 master mix for Forward reaction and the other for Reverse Reaction (account for one extra tube). E.g. You want 10 forward reaction samples, prepare for 11 samples…that would make:

SDW= 11ul x 11

Big Dye buffer= 4ul x 11

T7 Promoter (forward primer) = _2ul x 11

Total 187ul/11 = 17ul in each tube

*** Remember 2ul of BDT mix already aliquoted in tubes and 1ul DNA added last!

Similarly, prepare master mix for reverse reaction as well……..exact same except for the reverse T7 Terminator primer……

  1. Big Dye Terminator Mix is already prepared in aliquots
  2. Add DNA last in individual tubes

Purify Sequencing Reaction

  1. Take purification tubes out.
  2. Clean purification tubes 2X by adding 700 ul SDW and centrifuge 1 min @ max speed.
  3. Discard flow through and flip tube over to get rid of previous column residue.
  4. Get Sephadex (in 4˚C) and swirl it to mix. Add 700 ul Sephadex and centrifuge for 2 min @ 2 rpm! Sephadex form the column.
  5. Get one 1.5 microcentrifuge tube per each purification tube use and label accordingly.
  6. Insert the inner tube of the purification tubes into appropriate 1.5 microcentrifuge tubes.
  7. Spin sequence rxn product.
  8. Directly pipette each sequence rxn (~20 ul) product into appropriate column. Do not pour on top of broken column.
  9. Centrifuge at 2 rpm for 2 min.
  10. Save eluted sequence DNA(s) in 1.5 microcentrifuge tubes to dry in room N210 (Autoclave Room). Follow instruction on the wall. Let dry for 25 mins.
  11. Meanwhile, put the inner purification tubes back into the outer tubes and wash 2X with 700 ul SDW.
  12. Collect DNA samples in tube and:
  13. Give to Denise to sequence the DNA
  14. If she is not there, leave in -20˚C (your box) and give it to her when she is present.

Revision 01/20/07