Penn Genomics Analysis Core
Template Preparation & Purification Guidelines
Important: Criteria for cycle sequencing are more stringent than manual sequencing and many other molecular biological procedures. The success of sequencing reactions depends critically on having high purity template in the correct amount. The most important contaminants are bacterial proteins and cell wall carbohydrates, and organic solvents (ethanol, isopropanol, etc.). Please follow these recommendations for checking concentration and purity of your template DNA:
- Scan DNA sample in a spectrophotometer between 220 and 320 nm. If you do not have access to a scanning spec, measure absorbance at 220, 260 and 280 nm. In Figure 1 below, the solid line shows the absorption profile of pure DNA (RNA profile is very similar), and the dashed lines indicate how this profile is modified by the presence of contaminants. Nucleic acids have their peak absorbance at 260, with a 260/280 ratio of 1.8 for DNA, 2.0 for RNA. Estimate DNA concentration from OD260: conc (μg/ml) = OD260 X dilution factor X 50. Remember that OD260 can have contributions from DNA as well as contaminants.
Sample and primer concentrations and amounts for different types of templates are given below:
- Run a sample of DNA on an agarose gel with a quantitative standard (e.g. lambda HindIII digest, where amount of DNA in each band is known). By comparing intensities of template band with comparable size bands in standard lane, estimate concentration of DNA (see Figure 2 below). Compare this estimate to the OD260 estimate of DNA concentration. If OD260 estimate is significantly higher than gel estimate, it indicates contamination with RNA or RNA fragments. An RNase treatment, followed by phenol/chloroform extraction and precipitation would be recommended.
- If the scan profile or low OD260/280 ratios indicate protein/carbohydrate contamination, perform the phenol extraction and precipitation procedure described for optional cleanup below. If organic contamination is suspected (OD220>>OD260), you can usually remove them by precipitation and redissolving. Alternatively, small contaminants can be washed away by centrifugation through a microcon or centricon ultrafiltration concentrator.
|Template Type||Size & Template Amount/rxn||Template Conc / Vol||Primer Amount||Primer Conc / Vol|
|PCR||0.1-1 Kb; 10 ng per 100 bp||1.6 - 16 ng/μl x 6 μl||3.2 pmoles||1.1 μM x 3μl|
|Plasmid||2-15 Kb; 0.5 μg||80 ng/μl x 6 μl||3.2 pmoles||1.1 μM x 3μl|
|Phage/Cosmid/P1||20-100 Kb; 1.0 μg||110 ng/μl x 9 μl||12 pmoles||4 μM x 3μl|
|BAC||200 Kb; 1 - 2.0 μg||220 ng/μl x 9 μl||12 pmoles||4 μM x 3μl|
Table 1: Sample and primer concentration and volume
Figure 1: Spectroscopic analysis of DNA sample
Figure 2: Gel analysis of DNA sample
In this example 1 ul of lambda HindIII digest containing 500 ng DNA was loaded in the outer lanes, and 10 ul of sample was loaded in lanes 2 and 3. Since the lambda genome is 50 kb in size, the 4.4 kb band contains 500 X 4.4/50=44 ng of DNA. The sample in lane 2 is a little brighter, probably containing 80 ng of DNA. Therefore, DNA concentration in the sample is 8 ng/ul.
Grow 1 colony in 5-25ml LB + antibiotic overnight (~16hrs) at 37oC with shaking. Cell density should not exceed 1.5 A600 units. Debris from high density culture inhibits the reaction.
- Purification by Qiagen Columns
Use 1.5 ml of culture lysate per mini column, 5-10 ml per midi column, or 50-100 ml per maxi column. Follow Qiagen procedure, but add fresh RNase to 10U/ml (instead of, or in addition to, the RNase in the P1 buffer). Mix suspension very gently during and after cell lysis. Centrifuge lysate twice after lysis (supernatant should be clear). Treat with an additional 10U/ml RNase for 30min at 37°C before loading onto the column. Following isopropanol precipitation, redissolve DNA in water and precipitate with ethanol: add 1/10 volume 3M sodium acetate + 3 volumes absolute ethanol, chill on ice 30 minutes. Spin at 10,000 RPM for 20 min, 70% ethanol wash, dry, redissolve in water (salts inhibit sequencing reaction).
- Purification by Promega Wizard Mini Preps
Use 3ml of culture per mini prep and follow Promega procedure. Spin lysate twice. Be sure the supernatant is clear before adding resin. It is often necessary to concentrate eluted samples by ethanol precipitation or drying. Precipitation also improves quality of template and allows resuspension in water.
- Optional Purification
- RNase treatment, followed by phenol/chloroform extraction and precipitation improves template quality, but care must be taken to remove all organics. Most samples treated in this manner can be sequenced successfully. Incubate with 10 U/ml fresh RNase for 30 min at 37oC, extract with phenol (until interphase is clear), phenol/chloroform, then chloroform. Precipitate with ethanol, wash pellet with 70% EtOH, dry, and redissolve in water.
- Template quality may also be improved using a 100K centrifugal concentrator (minicon, centricon - dilute 5-10 μg of DNA to 50-100 μl with water and centrifuge through concentrator at 3000g for 10 min, add water and repeat 3-5X) or by dialysis for 2hr using an 100K MW cut off Millipore Minitan-S membrane (float the dry membrane shiny side down on sterile water and apply the 20 μl DNA sample).
- PEG precipitation is also helpful in improving quality of template. Add 0.25 vol 4M NaCl and 1.25 vol 13% PEG 8000, incubate on ice 20 min, pellet DNA, redissolve in water.
- Alternative Methods
Templates purified by phenol-chloroform extraction or CsCl gradients can be used, as long as sufficient further precipitations/washes are done to remove organics/CsCl.
If PCR product is a single band, purify with Qiagen or Wizard PCR purification columns. These columns use salts and ethanol, so extensive washing of pellet and complete drying of ethanol is necessary. Several washes with water through a microcon 100 concentrator can also be used for purification or clean-up. If there are multiple bands, run products on low melting point agarose gel, excise band, melt agarose (completely), purify DNA using Promega Wizard PCR preps following recommended procedure. Alternatively, run products on a gel, cut and electroelute desired band. Quantitate template on gel (OD260 can be misleading if salts/alcohols are left in sample). Submit 10ng of DNA / 100 bp / reaction in ≤ 5 μl water.
Lambda preps isolated by two consecutive bandings in CsCl, followed by RNase treatment and phenol-chloroform extraction, yield good DNA for sequencing. Purification by Qiagen Lambda prep columns also gives adequate results. Below is a procedure for preparing lambda DNA by extractions and precipitations.
- Place 200 μl of a healthy overnight culture of the appropriate host strain into a clear plastic, sterile 10 ml snap-cap tube. The choice of bacterial strain is important. Choose a host in which lambda replicates vigorously, e.g. use strain Y1090 for a lambda gt11 clone. If appropriate, include 0.2% maltose in culture medium to induce the lamB gene that codes for lamba receptors.
- Stab a well-separated plaque with a sterile toothpick and rinse the captured lambda phage particles into the cells aliquoted above. Alternatively, use 3x106 pfu of phage from a stock. Allow the phage to adsorb to the cells for 15 minutes at room temperature.
- Add 2 ml Tryptone Broth containing 10 mM MgSO4 and shake the culture at 37oC until the culture is lysed. Lysis is evident when the culture becomes clear and cellular debris is prevalent. This will take anywhere from 4 to 8 hours depending on both the host and lambda strain used. Add 10-100 μl chloroform and continue shaking to complete lysis if necessary. Pellet cellular debris at 10,000 rpm for 5 min.
- Pour the supernatant into a new 2 ml tube. Add 10 μl 2000U/ml RNase A and 5 μl 10 mg/ml DNase I. Incubate the sample at 37°C for 20 minutes.
- Add 0.12 g NaCl and invert the tube several times until the salt is dissolved, then add 0.2 g polyethylene glycol (8000 average MW) and invert the tube repeatedly until the PEG is dissolved. Let the sample sit at 4°C for 1 hr to overnight.
- Spin in a microcentrifuge for 20 minutes. Resuspend pellet in 0.5 ml SM (50 mM Tris, pH 8.0, 100 mM NaCl, 10 mM MgS04) and transfer to a 1.5 ml tube.
- Extract the sample once with chloroform. To the aqueous layer, transferred to a new tube, add: 40 μl 250 mM EDTA pH 8.0, 5 μl 20% sodium dodecyl sulfate, 2.5 μl of 10 mg/ml Proteinase K, and incubate the sample at 60°C for 30 minutes.
- Extract twice with phenol/chloroform, then twice with chloroform. Add 40 μl 3 M sodium acetate, pH 5.5 and 1ml 95% ethanol. Spin at room temperature for 10 minutes.
- Rinse the pellet with 750 μl cold 70% ethanol. Resuspend the DNA pellet in 400 μl distilled water.
- Add 200 μl 40% PEG8OOO, 30 mM MgSO4. Mix thoroughly, let stand at room temp for 20 min. Spin at room temperature for 20 minutes. Resuspend the pelleted DNA in 400 μl distilled water.
- Extract twice with chloroform. Add 40 μl 3 M sodium acetate and 1 ml 95% ethanol. Mix thoroughly. Spin at room temp for 10 min.
- Rinse the pellet with 750 μl cold 70% ethanol. Air dry pellet and resuspend in 50 μl distilled water. Yield is 1-2 μg.
BAC DNA prepared by CsCl gradient centrifugation, followed by Rnase treatment, extraction and precipitation work well as templates. Below is a modified alkaline lysis method that yields good quality DNA.
- Grow 200 ml of culture. OD 600 should be between 1 and 1.5.
- Spin down cells and resuspend in 15 ml of P1 adding fresh RNase (150 units).
- Add 15 ml of P2, mix by gentle inversion and incubate for 5 minutes at room temperature or until lysis looks complete.
- Add 15 ml of P3 and keep on ice for 15 minutes. Mix every 3 minutes by gentle inversion.
- Spin down cell debris 13,000 rpm for 30 minutes at 4°C. Spin a second time for 15 minutes to get rid of any debris still in suspension.
- Transfer supernatant to a fresh tube and precipitate with isopropanol. Wash pellet with 70% ethanol.
- Resuspend pellet in 1 ml H2O and add 0.25 ml 10 M NH4Ac to a final concentration of 2 M NH4Ac. Pellet precipitate by centrifugation at 13,000 rpm 30 min, keep supernatant.
- Transfer supernatant to a new tube and precipitate DNA with 2 volumes of ethanol. Pellet at 10,000 rpm for 15 minutes.
- Redissolve pellet in 2 ml of H2O and add 10 ml of QBT.
- Equilibrate QIAGEN tip 100 with 4 ml of QBT. Apply sample to column.
- Wash column with 10 ml of QC three times. Elute DNA with 4 ml of QF.
- Divide eluted DNA into 6 microfuge tubes, 0.67 ml each. Add 0.670 ml of isopropanol and chill on ice for 15 min.
- Spin DNA down at 10,000 rpm for 15 min and wash pellet with 70% ethanol as before.
- Dissolve in water after air drying. Yield is 5-8 μg.
Generally follow protocol given by ABI for Taq FS and dye terminator chemistry. Consult ABI protocols or facility staff for other chemistries. Typically, template is in 6 μl volume, primer in 3 μl, and reaction mix with Taq polymerase in 8 μl. Add DMSO to 5-10%, and double Taq concentration, for GC rich or difficult templates. Total reaction volume is 20 μl. The table below shows template, primer and reaction condition requirements for different templates when using dye terminators and Taq FS:
|Template Type||Template Amount||Primer Amount||# Cycles|
|PCR product (100 bp - 2 kb)||10ng/ 100 bp||3.2 pmole||25|
|Plasmid (2-15 kb)||0.35 μg||3.2 pmole||25|
|Phage, cosmid (20-50 kb)||1.0 μg||25 pmole||30|
|P1 (100 kb)||1.0 μg||25 pmole||30|
|BAC (200 kb)||2.0 μg||25 pmole||30|
Table 2: Dye terminated cycle sequencing reaction conditions
Reaction products are purifed with a Centrisep spin column (Princeton Seperations), or a Sephadex G-50 Quick Spin column from Boehringer-Mannheim, or CentriSep column washed and repacked with G-50. Currently the purification is done by ABI BigDye Xterminator reagents.
- CentriSep: Swell dry gel with 0.75 ml of water for 30 min. Spin in fixed angle microfuge rotor at 750g (~3,000 rpm) for 2 min. Put tubes in with mark oriented up, so that you can perform second spin in same orientation. Load sample in center of gel, repeat spin with tube in same orientation as the first spin.
- BMB Spin Columns: If using an angle rotor, orient the columns the same way for both spins. After the first spin, the gel material within the column will pull away from the column walls. Be sure to load your sample in the center of the gel material.
Penn Genomics Analysis Core is an Abramson Cancer Center Shared Resource that is approved and partially funded by the National Cancer Institute.