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Application Notes

Introduction:

Users of Big Dye version 3 chemistries have reported occasional dye blobs on sequence images even after cleanup of reactions using spin columns or plates. The blobs are apparently due to aggregation of the dyes. ABI has released a modification of the cleanup protocol supplied with the version 3 chemistry. The recommendation is to add 1/10th volume of 2.2% SDS to the sample, mix, heat to 95 degrees C for 5 minutes, cool to ambient temperature and spin briefly to collect sample in bottom of tube.. The sample is then added to the spin column as previously described. This additional step is also recommended for samples processed using Centri·Sep 96 plates.

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Nucleic acid hybridization with labeled probe sequences is a well known analytical tool used in molecular and cell biology. The utility of hybridization has been greatly increased by the development of in vitro methods for incorporation of label (either isotopic or nonisotopic) into the nucleic acid probe molecules. One of these methods is nick translation, where nucleotides are incorporated into double stranded DNA by DNA Polymerase 1 at sites where the molecule has been nicked by DNAse 1. Nucleotide mixes containing a label 32P-dATP or Fluorescein-dUTP) provide the pool of nucleotides which DNA Polymerase 1 will incorporate into the double stranded DNA molecule. The excess labeled nucleotides are then separated from the finished, "labeled" DNA.

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Introduction:

The high salt concentrations present in ligation mixes are known to reduce electroporation transformation efficiency. Recommendations to reduce salt concentration prior to electroporation include float dialysis, centrifugal ultrafiltration, and dilution. Each of these are either time consuming, not completely effective (reducing the transformation efficiency), or worse, result in significant DNA loss.

A Centri-Spin 20 protocol for desalting ligation reactions eliminates salts and co-factors while recovering >85% of the DNA. Electroporation transformation efficiencies are increased 2-fold.

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Introduction:

We have developed a spin column miniprep protocol for the purification of BAC DNA. No organic extractions are required. Typical yields between 0.6 and 1.0 µg are observed from an overnight 3-5 mL culture. A discussion of the data and results depicting the quality of BAC DNA purified is presented. Alternative protocols for preparation of BAC DNA from 25-250mL cultures are also presented. Observations of some factors that affect BAC yield will be discussed.

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BAC DNA

Prepare a solution of 0.2N NaOH; 1% (w/v) SDS and substitute this for the current lysis buffer and follow the protocol as normal.

Because Cosmids are under 80kb (actually under 60kb) they don't require the modified BAC Lysis buffer in our kit. You could still use it; however, we've found standard lysis buffer improves the yield.

Be sure that the lysate was diluted as instructed in Step 7. The PSI Pressure Tip (included in the bag of columns) can be used to initiate or assist flow. Place the Pressure Tip on the end of a 3 mL Luer Lock syringe. Pull back the syringe plunger and place the tip on the top of the column. Depress the plunger to start flow. If flow doesn't start reseat the tip on the column. Maintain a flow rate of 1-2 drops/second until all of the lysate has been expelled from the column.

Do not release the pressure on the plunger while the syringe is seated on the column; If released the resulting vacuum may disrupt the column bed.

This procedure may also be needed to assist flow during the wash steps.

Big BAC

Yes. The columns may simply be rinsed well with reagent grade water and reused.

Centri·Sep
Centri·Sep 8

Centrifuges equipped with 96-well plate carriers may be used to process Centri-Sep 8 strips

Labeling Kits

No. The fluorescent dyes (FAM, TAMRA and ROX) provided with the kits are activated as NHS (N-Hydroxy-Succinamide) esters. NHS esters are reactive with amines. Since Tris is an amine, it will react with the activated dye and act as a scavanger during the labeling reaction. This will reduce the efficiency of the labeling reaction. It is recommended that you exchange your protein into a PBS buffer prior to the labeling reaction using a Centri-Spin spin column (available from Princeton Separations) or related method based on gel filtration. The protein can be exchanged back into the Tris buffer following the labeling reaction.

We typically obtain greater than 90 percent recovery of labeled protein from our spin columns.

Pro·Spin

Yes. However, the conditions for purification will vary depending upon the properties of the protein. Recovery of certain proteins may be affected due to hydrophobic or ionic interactions. This must be considered when designing the pH, salt concentration and ionic strength of the hydration buffer. Non-ionic detergents may be necessary for many membrane proteins. Proteins can also be degraded by proteases. Protease degradation can reduce recovery from the column. Appropriate protease inhibitors must be added to the hydration buffer to improve recovery of intact proteins.

Endopeptidases

You can store the reconstituted enzyme in the freezer. You can freeze/ thaw about 5-6 times.

–10°C or –20°C is fine. It is acceptable to freeze at –70°C and –80°C.
If using a frost free freezer put the samples in the middle of the freezer not on the door.

Since the enzyme is modified we do not recommend it be stored at ph 3.0, this will inactivate the enzyme. We recommend storing the enzyme at a pH between 6-8.

You can reconstitute with water or any buffer, preferably Tris-HCl pH 8.0.

The enzyme can be reconstituted in H20 or buffer up to 100µl.

Tech Tips

Centri-Sep 96 filter plates have been developed and optimized to remove the excess dye terminators from the extension products in the Big Dye®Terminator cycle sequencing reactions. Sample volumes of 5 to 20 microliters are efficiently purified in less than 10 minutes. Purified samples result in clean electrophoretic results and long base reads. Transferring the samples to the plates is an extremely important part of the procedure and is essential to getting good quality sequencing results.

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Introduction:

Reduced recovery of protein samples may occur when using Centri-Spin spin columns due to specific or non-specific interaction with the column matrix. Below is a list of common modes of interaction and suggestions for minimizing their effect on sample recovery.

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Introduction:

CENTRI-SEP 96 multi-well plates are an excellent solution to the difficulties encountered in removal of excess dye labeled terminators from dye terminator reactions. The hydrated matrix in these plates has been optimized for use with Big Dye reactions. When properly used, CENTRI-SEP 96 plates will yield sequencing reactions equivalent in cleanliness to single CENTRI-SEP spin columns. Gel images obtained using reactions prepared with CENTRI-SEP 96 plates will be free of interfering smears, blobs, and gel haze caused by the presence of excess dyes. Sequences may be read from the first base and longer read lengths may be obtained due to reduced background. These improved reads result in a lower cost per base than less effective precipitation methods.

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Introduction:

CENTRI-SPIN spin columns provide a convenient and rapid method to remove excess label from nick translation or PCR labeling reactions (see Application Note A-1). For example, CENTRI-SPIN-20 will remove up to 99% of free label from a nick translation reaction in a simple 2 minute centrifugation. CENTRI-SPIN columns are not routinely tested for the presence of RNase and the majority of applications have been developed for proteins and DNA. However, the spin columns may also be used for RNA labeling reactions if precautions are taken to reduce the possibility of RNase contamination. Some of these precautions are discussed below.

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Online Tools

Note: The rotating speed in Revolutions per Minute (RPM) and the rotating radius in centimeters.

Calculate RPM from Radius and RCF

radius:  
rcf:
 

Note: Measure the rotating speed in Revolutions per Minute (RPM) and the rotating radius in centimeters.

Calculate RCF from Radius and RPM

radius:  
rpm:
 

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