Centricon microconcentrators concentrators

For some RNases, especially recombinant RNases, there can be a large loss of protein after concentration with a Centricon microconcentrator. Other methods of concentration, such as Diaflo ultrafiltration (Amicon Inc.) using a YM3 membrane, have not been successful in these cases. This concentration step can be avoided by either dialyzing as described in Note 1 or by starting with more material, such as 1 mL of a 10-20 mg/mL RNase solution. The final protein concentration should be 6.4 mg/mL. 

An F/P ratio of two to five is optimal, since ratios below this yield low signals, whereas higher ratios show high background. If the F/P ratios are too low, repeat the coupling reaction using fresh fluorochrome solution. The IgG solution needs to be concentrated prior to reconjugation (e.g., Centricon-30 microconcentrator from Amicon Co., Beverly, MA, can be used to concentrate the IgG solntion). 

A DNA extraction protocol that has proved useful for most ancient tissues is a modification of the protocol initially published by Blin and Stafford.20 Approximately 0.1 g of small pieces of soft tissue is added to 5 ml of extraction buffer containing 10 mM Tris-HCl (pH 8.0), 2 mM ethylenediaminetetraacetic acid (EDTA), 10 mM NaCl, 1% (w/v) sodium dodecyl sulfate (SDS), 10 mg/ml dithiothreitol (DTT), and 0.5 mg/ml proteinase K. Incubation at 37° with gentle agitation overnight will allow most or all of the tissue to go into solution. An equal volume of phenol, equilibrated with 1 M Tris-HCl (pH 8.0), is added. When the phenol is being equilibrated, care should be taken to use uncontaminated Tris buffer and to measure the pH only on aliquots that are removed from the water phase and then discarded. Two phenol extractions and one chloroform extraction are performed, and the water phase is concentrated and purified on a Centricon 30 microconcentrator (Amicon, Danvers, MA). The reten-tate can be stored frozen, preferably in a few aliquots. In all cases solutions should be manipulated with DNA-free positive displacement pipettes. 

Prepare cDNA from poly(A) RNA with MMLV reverse transcriptase (100 p.g of RNA should yield about 50 g,g of cDNA). Wash the cDNA extensively with TRES (10 mM Tricine, pH 6.8, 0,4 mM EDTA, 0.1% SDS RNase-free) in a Centricon-30 microconcentrator (Section 3.1.4.2). Recover RNA/DNA (about 50 nD, save 2% of the sample and concentrate the rest in a SpeedVac to 15-20 p.1. 

Concentrate the pooled RNase solution to 0.5 mL using a Centricon P-3 microconcentrator. Determine the final volume and concentration of the solution as described in step 2 (see Note 2). 

The concentration of the antibody solution should be at least 4 mg/mL. Concentration by Centricon P30 microconcentrator may be required to achieve this (see Note 7). 

The reaction may be concentrated with a Centricon P30 microconcentrator before application to the sizing column to reduce the number of chromatographic columns that must be performed. Before concentration, however, an analytical run of the reaction before and after concentration should be performed to ensure that the concentration step does not result in an increase of higher-mol-wt aggregates. We find that some RNase-antibody conjugates can not be concentrated without a loss (in some cases up to 50%) of conjugate. 

At this stage, the pooled RNase-antibody reaction may be concentrated on a Centricon P30 microconcentrator, however, the conjugate should be concentrated with caution because some RNase-antibody reactions will result in as much as a 50% loss of material as a result of aggregation. 

Fraction VI is concentrated and equilibrated with buffer A containing 0.6 M NaCl on an Amicon (Danvers, MA) microconcentrator (Centricon 30). This fraction is loaded on a 5-20% sucrose gradient in buffer A containing 0.6 Af NaCl and centrifuged at 175,(KX)g for 40 hr at 4° in a SW 41 Beckman rotor. Fractions of 350 p,l are collected. Active fractions are pooled, concentrated, and finally equilibrated with 25 mAf NaH2P04/Na2HP04, pH 7,0.5 mAf DTT, 0.5 mAf EDTA, and 100 mAf NaCl (fraction VII, 0.36 ml, 0.08 mg of proteins). 

To Prepare DNA for Sequencing To purify DNA for sequencing, the reaction mixture can be concentrated (to approx. 30-40 pi) using a Centricon 30 microconcentrator (Amicon, Danvers, MA) or an Ultrafree Microcentrator 30,000 (Millipore, Bedford, MA) which remove salts, excess nucleotides and primers. Both methods retain more DNA than ethanol precipitation and the latter is less expensive, faster to use, and works well. In our laboratory... 

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