Once the template is prepared, be sure to check the DNA quality before submitting it for sequencing. Quality can be determined by:
- Agarose gel electrophoresis - Purified DNA should run as a single band on an agarose gel (Uncut plasmid DNA can run as 3 bands: supercoiled, nicked, and liner). Contaminating DNA or RNA will show up in the gel.
- Spectrophotometry - The A260/A280 ratio should be between 1.7 and 1.9. Smaller ratios usually indicate the presence of contaminating proteins or phenol. The NanoDrop ND-1000 spectrophotometer takes 1-2 µL sample volumes and can detect concentrations from 1.5 - 3700 ng/µl without dilutions.
NOTE: Neither method will show the presence of contaminating salts.
- Qiagen Plasmid Mini/Midi/Maxi Kits or QIAprep® Spin Miniprep Kit
- Promega Wizard® Plus SV Miniprep (Do not use earlier versions of Wizard® Plus Minipreps)
- Bio-Rad Quantum Prep® or AurumTM Plasmid Mini Kit
- Eppendorf Perfectprep® Plasmid Mini
- GE Healthcare TempliPhi™ DNA Amplification Kit
- Modified Alkaline lysis/PEG precipitation method
Helpful Hints for Plasmids
- Be sure to restreak bacterial colonies to make sure one colony is isolated with the correct plasmid + insert.
- Supply purified DNA in water (10mM Tris is also OK, but not TE because the EDTA can interfere with the sequencing reaction).
- For best results, use EndA- strains of E. coli to propagate plasmids, such as DH1, DH5a, C600, or XL1-Blue. The endonuclease activity can greatly reduce DNA quality.
- Do not use E. coli strains that produce excess amounts of carbohydrates such as HB101, TG1, or JM series.
- Use high-copy-number plasmids when possible.
- Do not overgrow bacteria because the cells will lyse, releasing degraded bacterial genomic and plasmid DNA.
- Use of Luria-Bertani (LB) media is recommended for bacterial growth. Richer media, such as Terrific Broth (TB) can lead to very high cell densities which can overload columns or membranes used for plasmid purification.
- Never vortex DNA during the purification process. This can shear the chromosomal DNA, making it impossible to separate the chromosomal from the plasmid DNA.
ABI's Modified Alkaline Lysis/PEG Precipitation Procedure
- Deionized Water
- Ethanol, 70%
- GET Buffer (50mM glucose, 10mM EDTA, 25mM Tris, pH 8.0)
- Isopropanol, 100% (anhydrous)
- PEG 8000, 13% (sterilized by autoclaving, rather than by filtration)
- Potassium Acetate, 3M, pH 4.8
- RNase A (DNase-free), 10mg/mL
- Sodium Chloride (NaCl), 4M
- Sodium Hydroxide (NaOH), 0.2N, with 1% SDS (freshly made)
NOTE: To minimize shearing of contaminating chromosomal DNA, do not use a vortexer during this procedure.
- Pellet 1.5-mL aliquots of culture for 1 minute in a microcentrifuge at maximum speed. (NOTE: A total culture volume of 4.5 mL can be spun down per tube without changing volumes in the procedure. This allows you to achieve a threefold increase in yield while eliminating the need for extra tubes and additional handling.)
- Remove the supernatant by aspiration.
- Resuspend the bacterial pellet in 200 µL of GET buffer by pipetting up and down.
- Add 300 µL of freshly prepared 0.2 N NaOH/1% SDS. Mix the contents of the tube by inversion. Incubate on ice for 5 minutes.
- Neutralize the solution by adding 300 µL of 3.0 M potassium acetate, pH 4.8. Mix by inverting the tube. Incubate on ice for 5 minutes.
- Remove cellular debris by spinning in a microcentrifuge at maximum speed for 10 minutes at room temperature. Transfer the supernatant to a clean tube.
- Add RNase A (DNase-free) to a final concentration of 20 mg/µL. Incubate the tube at 37°C for 20 minutes.
- Extract the supernatant twice with chloroform.
- Add 400 µL of chloroform.
- Mix the layers by inversion for 30 seconds.
- Centrifuge the tube for 1 minute to separate the phases.
- Transfer the upper aqueous phase to a clean tube.
- Add an equal volume of 100% isopropanol. Mix the contents of the tube by inversion.
- Spin the tube in a microcentrifuge at maximum speed for 10 minutes at room temperature.
- Remove the isopropanol completely by aspiration.
- Wash the DNA pellet with 500 µL of 70% ethanol. Dry under vacuum for 3 minutes.
- Dissolve the pellet in 32 µL of deionized water.
- Add 8.0 µL of 4 M NaCl, then 40 µL of autoclaved 13% PEG 8000.
- Mix thoroughly, then leave the sample on ice for 20 minutes.
- Pellet the plasmid DNA by spinning in a microcentrifuge at maximum speed for 15 minutes at 2-6°C.
- Carefully remove the supernatant. Rinse the pellet with 500 µL of 70% ethanol.
- Resuspend the pellet in 20 µL of deionized water. Store at -15 to -25°C.
Helpful Hints For PCR Products
- If more than one product is present, either the PCR conditions must be reoptimized or the reaction must be gel purified to isolate the desired product.
- Excess PCR primers and dNTPs must be removed prior to sequencing. The primers will compete with the sequencing primer, resulting in a noisy read with overlapping peaks. Excess dNTPs can cause an imbalance in the sequencing reaction. PCR protocols using limiting amounts of primers and dNTPs, so that most of the primers and dNTPs are exhausted during the reaction, can be sequenced directly without purification as long as only one product is formed.
- Supply purified PCR product in water (10mM Tris is also OK, but not TE because the EDTA can interfere with the sequencing reaction).
Shrimp Alkaline Phosphatase/Exonuclease I Treatment
The SAP/Exo I treatment degrades nucleotides and any single-stranded DNA (primers) left over after the PCR. This is the protocol recommended by ABI. **Other protocols suggest a shorter incubation time because the enzyme will start degrading the PCR product if allowed to react for too long.
- For each sample combine the following
- SAP (1 Unit/µL), 2 µL
- Exo I (10 Units/µL), 0.2 µL
- Deionized water, 6.0 µL
NOTE: This procedure works well using 0.5 units of each enzyme per microliter of PCR product used
- Add 4.0 µL of PCR product to the above mixture
- Incubate at 37°C for 1 hour**
- Incubate at 72°C for 15 minutes to inactivate the enzymes
BAC™s, YAC™s, PAC™s, & Cosmids
- Qiagen-tip 100/500 kits.
- Other types of kits/methods may also work. Those suggested have been successfully used by Core Facilities' customers.