At this stage your clones have been PCRed and run out on a gel to check the quality of inserts and the clones selected for sequencing regridded to a fresh microtitre plate. The PCR products now have to be cleaned (excess dNTPs and PCR primers must be removed) before they can be used for sequencing.

We have sucessfully used Shrimp Alkaline Phosphatase (SAP)¹ and Exonuclease I (EXO I)² to prepare our PCR products for sequencing. SAP removes the phosphate groups from the excess dNTPs left over from the PCR reaction and Exonuclease I digest the single stranded PCR primers into dNTPs and the phoshate groups are removed by the SAP.

Both enzymes are readily inactivated by heating at 80^oC 10min so do not effect the sequencing reation mix.

To 15ul of PCR product we typically add:

1ul of neat SAP (1 unit/ul)

1.5 ul 1/10 diluted ExonucleaseI (1 unit/ul)

These enzyme concentraions and length of incubation may need to be adjusted to suit your PCR reaction mix (see manufacturers instructions)

Protocol for processing 96 well PCR plates of regridded PCR products:

All stages should be carried out on ice

(1) 96 well PCR plate containing 15ul PCR product of each clones selected for sequencing.

(2) For each clone you need

1ul of neat SAP (1 unit/ul)

1.5 ul 1/10 diluted ExonucleaseI (1 unit/ul)

To clean 96 clones prepare enough master mix for 112 reactions

Master mix (prepare on ice)=

112 ul neat SAP (1u/ul)

168 ul 1/10 diluted ExonucleaseI (diluted to 1u/ul in the SAP dilution buffer provided with the SAP)

Both enzymes can be mixed together to final volume 280ul MM

(3) Add 2.5 ul of master mix to each well of the PCR plate. Ensure you mix the enzymes in well by gentle pipetting. Take care not to cross contaminate your wells or master mix.

(4) Seal plate with sticky tape sealer (no oil is needed if hot lid used on PCR machine)

(4) On PCR machine heat to

• 37^oC 30 min
• 80^oC 10 min
• cool to 4^oC

(Cycle 36 on our hybaid touchdown)

(5) The 17ul volume PCR product is now ready for sequencing or can be stored frozen until needed. Typically we use 5ul of cleaned PCR product per 1/2 sequencing reaction.

Protocol for sequencing SAP/EXOI cleaned PCR Products in 96 well PCR plate

 We usually prepare 1/2 ABI sequencing reactions.

For each 1/2 sequencing reaction:

5ul SAP/EXOI clean PCR product

1ul 1.6pmol/ul sequencing primer

4ul dye terminators³

All steps to be carried out on ice

(1) Prepare enough master mix of sequencing reaction for 96 clones

master mix for 112=

112ul 1.6pmol/ul primer

448 ul dye trminators

560 ul master mix

(2) Aliquot 5ul of sequencing master mix to each well of a fresh PCR plate.

(3) Transfer 5ul of each 17ul of clean PCR product to the corresponding location in the PCR plate containing sequencing mix. Mix PCR product and seqeuncing reaction well by gentle pipetting. take care not to cross contaminate wells.

(4) Add one drop of oil to each of the wells and seal plate (oil not necessary if hot lid used)

On PCR block (program 37 on our touchdown)

• 95^oC 30 sec
• 50^oC 20sec
• 60^oC 4min

25 cycles

• 4^oC 30min

hold 4^oC

(Cycle 3 on our Touchdown machine)

(5) The sequencing reaction products must be precipitated individually. For each 10ul sequencing product you need

2ul 3M Sodium Acetate ph5.2

50ul 100% ethanol (-20^oC)

Prepare a master mix enough for 96 reactions

Master mix for 110 reactions=

220ul 3M Soduium acetate

5500ul 100% Ethanol (-20^oC)

5720ul MM

Aliquot 52 ul of Ethanol/Soduium Acetate to 96 clean, labelled 1.5 ml (or 0.5ml) eppendorffs.

(6) Remove the sequencing reaction product from underneath the oil and transfer to the relevant tube containing Ethanol/Soduium Acetate

(7) Precipitate the DNA for 10 min on ice (or longer -20^oC)

(8) Pellet DNA by centrifugation at 13K 4^oC 30min

(9) Remove the ethanol taking care not to disturb the pellet. Note a pellet is not always seen so take care to align eppendorffs in centrifuge so you know where to expect the pellet to be.

(10) Gently add 250ul 70% ETOH (-20^oC)

(11) Centrifuge again 13K 4^oC 10min

(12) Remove the 70% ETOH taking care not to disturb the pellet

(13) Pulse spin again to pellet drops of ethanol from sides of tube and remove final few ul of ethanol

(14) Allow pellets to air dry. It is important to remove all ethanol from the pellet.

Store dry pellets at -20^oC until loading on sequencing gel.

For loading on a gel pellets should be resuspended in 3ul freshly prepared loading buffer

Loading buffer=5ul deionised formamide to 1ul dextran blue.

Resuspend pellet vigorously.

heat to 90^oC for 2min and place on ice

load 1.5 to 1.8ul

(1) Shrimp Alkaline Phosphatase (SAP) Amersham E 70092Z (1000 units, 1u/ul)

(2) Exonuclease I (EXO I) Amersham E70073Z (2500 units, 10u/ul)

(3) ABI

dRhodamine Terminator Cycle Sequencing Ready reaction= cat no 403042 (100 full reactions)

Dye Terminators Cycle Sequencing ready reaction= cat no 402079 ( 100 full reactions)