SUPPORT

Please consult our F.A.Q. list below, or submit your own question or request

Application Notes

  • ABI releases revised spin column protocol for difficult samples when using Big Dye v. 3.0.

    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.

  • Effective Removal (>99.99%) of free Nucleotides from Nick Translation Reactions using Centri-Spin 20

    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.

  • Improving Electroporation Transformation Efficiencies using Centri-Spin 20 separation columns.

    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.

  • Procedures for the Purification of Bacterial Artificial Chromosome (BAC) DNA.

    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.

BAC DNA

  • Can our kit be used for Cosmid purification?

    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.

  • Sometimes the liquid flow through the column in the BAC kit (Cat. No. PP-120) is very slow. What can I do to speed it up?

    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

  • Can the columns supplied with the Big BAC kit be reused?

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

Centri·Sep

  • Can I use Sep columns to purify RNA probes? What can I resuspend in?

    Yes, but we don't guarantee they are RNASE free. You can resuspend in DEPC(inactivates RNASE) or use RNASE Zap.

  • Can I use the Centri-Sep columns to clean-up a genomic prep?

    No. It will definitely clog up your column.

  • The protocol for Centri-Sep columns says "can be used with ABI model #'s 373A or 377A sequencer", can they be used with the ABI 310 capillary system?

    Yes they can.

Centri·Sep 8

  • Do I need a special rotor to centrifuge Centri-Sep 8-well strips?

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

Labeling Kits

  • Can you use Tris buffers when labeling proteins with FluoroSpin 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.

  • What yield of labeled protein can I expect using your protein labeling kits?

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

PCR 96

  • Is there EDTA in any of the PCR buffers?

    No.

  • We are using plates for microarrays…Is there anything that will fluoresce in the elution buffer ?

    No.

  • What is the vacuum manifold we use?

    Polyfiltronics Univac#900101(vacuum is Curtin Matheson Scientific Inc.# 5KH36KN90)

Pro·Spin

  • Can Pro-Spin be used for purification of proteins other than antibodies?

    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

  • After reconstituting the enzyme can it be stored in the freezer?

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

  • At what temperature should the reconstituted enzyme be stored?

    –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.

  • Should the enzyme Lysine C be stored at pH 3.0?

    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.

  • Should you store the frozen product in plastic or glass?

    Either is acceptable.

  • The insert suggests reconstituting with deionized water. Is there any other appropriate buffer?

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

  • The product insert recommends you recon with 20 ul can you use more?

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

Sep 96

  • Can Sep 96 plates be used with a vacuum manifold instead of a centrifuge?

    No.

Tech Tips

  • Manual Sample Loading Techniques for Centri-Sep 96 filter plates

    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.

  • Maximizing sample recovery when using Centri-Spin columns for desalting Proteins.

    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.

  • Use of CENTRI-SEP 96 filter plates for cleanup of Dye Terminator Sequencing Reactions

    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.

  • Using CENTRI-SPIN columns with RNA Samples

    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.

Online Tools

  • Calculate RPM from Radius and RCF

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

    Calculate RPM from Radius and RCF

    radius:  
    rcf:
     
  • Calculate RCF from Radius and RPM

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

    Calculate RCF from Radius and RPM

    radius:  
    rpm: