PCR Reaction Mix: (50 m L)  

• DNA template: 1 m L
• Forward primer: 3 m L Tm =
• Reverse primer: 3 m L Tm =
• Pfx or Taq polymerase: 0.5 m L Predicted product:
• Pfx or Taq buffer: 5 uL time ext.=
• dNTPs: 1 m L PCR Cycle:
• MgSO 4 (only for Pfx) (1 m L)
• H 2 O: 36.5 m L
• TOTAL: 50 m L

Restriction Digest Mix: (25 m L)

• DNA target: (at least 1000 ng; max. 10 m g) variable Incubation usually at 37 ° C, 1.5-2 h
• Enzyme Buffer: (10X) 2.5 m L Inactivation at 65-80 ° C for 20 min.
• BSA: (100 X) (not always required) 0.25 m L
• Restriction enzyme: 1 m L (usually 5-10x excess)
• H 2 O: to volume
• TOTAL: 25 m L

Shrimp Alkaline Phosphatase (SAP) Mix: (30 m L)

• DNA target: 25 m L Incubation at 37 ° C for 1 hr.
• SAP: 1.5 m L Inactivation at 65 ° C for 20 min.
• SAP buffer: (10X) 3.0 m L
• H 2 O: 0.5 m L
• TOTAL: 30 m L

  CIP (Calf Intestinal Alkaline Phosphatase) Mix:

•   DNA target: variable, max 10 m g
• CIP 0.5 units/ m g DNA; DO NOT USE EXCESS!
• CIP buffer (or NEB buffers 2, 3, or 4; 10X) 5 m L
• H 2 O: to volume m L
• TOTAL: 50 m L

Inactivate by running on agarose gel and extracting with Minelute/Qiaquick Qiagen kits.

Also, inactivation is possible by adding EDTA (10mM) and incubating 10 min. at 75 deg. C, but it may be unreliable.

DNA Ligation Mix: (20 m L)

• DNA insert: X m L (DNA concentration of X:Y is 3:1)
• Plasmid backbone: Y m L (use 50ng vector)
• DNA ligase buffer: (10X) 2 m L Incubate at 16 ° C overnight OR 2h @RT
• DNA ligase: 1 m L Also may run with PCR machine
• H 2 O : to volume (ana -> ligation)
• TOTAL: 20 m L

Calc. formula: (50ng vector x insert size (kb) x insert:vector ratio)/Vector size(kb) = ng insert

Ethanol Precipitation:

• •  Add 1 / 10 vol. of 5M ammonium acetate and vortex.
• •  Add 1.5 m L DNA pellet paint.
• •  Add 2 vol. of ice cold ethanol and vortex.
• •  Store at -80 ° C for 1 hour.
• •  Spin at 12,000 rpm at 4 ° C for 15 min.
• •  Remove supernatant.
• •  Add 100-200 m L 70% ethanol.
• •  Spin at 4 ° C for 5 min.
• •  Repeat steps 6-8 again.
• •  Remove supernatant.
• •  Allow to air dry or dry under vacuum.
• •  Resuspend DNA in H 2 O. (usually 10 m L, to concentrate.)

Plate Pouring:

• Thaw agar on hotplate and allow it to cool (approx. 1 hr.)
• Add appropriate antibiotic. (Ex. 50 mg of ampicillin and allow to mix.)
• Pour plates, cover, and allow to stand until the agar has solidified.
• Stack, leave at room temperature for several hours.
• Store at 4 ° C.

Transformation:

• •  Take 50 m L aliquot of SS320 cells (or electrocompetent DH5 a ) and place on ice along with cuvette.
• •  Add 1-3 m L of DNA (ethanol precipitated) and pipette-mix.
• •  Transfer cells to cuvette and electroporate (zap with machine). Note time constant.
• •  IMMEDIATELY add 950 m L LB media to cells, pipette-mix and transfer culture to 1.5ml Eppendorf tube.
• •  Incubate Eppendorf tube at 37 o C (1/2 hr for Amp R , 1hr with shaking for Kan R )
• •  Plate 100 m L aliquots on LBA + antibiotic (usually Amp) plates. For circular plasmids (stock vector) use 10 m L aliquots.
• •  Store plates in incubator at 37 o C, O/N (<16hrs for Amp, as Ampicillin gets extruded in the media by resistant bacteria, so will get lots of mini-satellite colonies after 16 hrs).
• Note: can store culture plates at 4 o C for ~ 6 mths.

Target annealing protocol:

1. Reconstitute oligos in H 2 O
2. Measure OD and prepare 5pmol dilution
3. Make annealing mix:
   • Annealing reaction mix:

• 2 m l 10X STE buffer
• 9 m l fwd strand
• 9 m l rev strand
• -------
• 20 m l total volume

4. Incubate mix in boiling water bath (100 o C), 2 min.
5. Remove samples with 250 ml of H 2 O and transfer to ice bucket.
6. Allow to cool slowly at RT.

Note : Do ethanol precipitation on above reaction (regular protocol), but in case of storage, add 2 m l glycogen before freezing at -80 o C to increase DNA recovery.

AquaGenomics (cat # 2030) Drosophila DNA extraction protocol

( http://www.aquaplasmid.com/)

1. Add 50 ul AquaGenomics = AG lysis solution to fly in 1.5 ml microfuge tube (1 fly; for 3 flies/tube, use 100 ul lysis solution)
2. Homogenize with Kontes disposable pestle, either by hand or with the Kontes pellet pestle motor for 5-10 sec. Up to 48 preps at a time can be done. Leave the homogenized flies on the desk until all flies have been homogenized.
3. Incubate at 60° C for 4 min.
4. Vigorously vortex each tube 20-40 sec.
5. Centrifuge sample at max. speed (16000 rpm) for 4 min.
6. Transfer supernatant to new tube containing one volume isopropanol. Mix.
7. Centrifuge at max speed 4 min; decant supernatant.
8. Rinse with ~500 µl 70% ethanol by shooting the ethanol solution from a squeeze bottle just below the rim of the tube. Decant ethanol and pipet off remaining solution.
9. Allow tubes to dry ~ -15 min at room temperature.
10. Add 30 ul 10 mM Tris, TE, or ddH 2 O to DNA. Leave on bench overnight, vortex, or heat at 60° for one hour to resuspend.
11. Store at either 4° or -20°C

.5µl of this solution is sufficient for a PCR reaction.

Notes for the user:

There is a layer of greasy stuff that forms at the top of the solution after centrifugation-although we try not to transfer this, we often end up getting some of it-even if we use a larger volume. This pellets with the DNA after the Isopropanol wash, but doesn't seem to affect the quality of the DNA. We use it for PCR, and even several months later, get good results even with multiple freeze-thaws or storage at 4°. I suspect if I was wanting to archive this DNA for years, I would use more solution and do a second spin, or remove the final solution to a new tube after resuspending the DNA and spinning again. I have never measured the amount of DNA obtained, but I could if anybody needs that number. Also, our rather insane grad student wants to add that she has done 96 at a time at room temp with good results.