High Throughput Reaction Assembly

Abstract

Provided herein is a reverse transcriptase mixture comprising a reverse transcriptase and a colored dye at a concentration in the range of 0.003%-1% (v/w). The colored dye may be visually observed during transfer of the mix from one vessel to another and addition of the mix to another mix can be confirmed by eye by observing the colored dye.

What is claimed is: 
     
         1 . A reverse transcriptase mixture comprising:
 (a) a reverse transcriptase; and   (b) a colored dye at a concentration in the range of 0.003% to 1% (w/v).   
     
     
         2 . The mixture of  claim 1 , wherein the colored dye is one or a combination of xylene cyanol, tartrazine, orange G or a combination of two or more of xylene cyanol, orange G and tartrazine. 
     
     
         3 . The mixture of  claim 1 , further comprising an inert high-density material. 
     
     
         4 . The mixture of  claim 3 , wherein the inert high-density material is glycerol or an equivalent selected from the group consisting of Glycerol, Ficoll, Polyethylene Glycol, Dextran or Polysucrose and the concentration of the glycerol or equivalent is in the range of 5% to 80% v/v. 
     
     
         5 . The mix of  claim 1 , wherein the reverse transcriptase is at a concentration in the range of 2-400 units/ul. 
     
     
         6 . The mixture of  claim 1 , further comprising one or more components selected from dNTPs, nucleic acid primers, RNase inhibitors and DNA adaptors. 
     
     
         7 . A kit, comprising: a reverse transcriptase and a colored dye, wherein the reverse transcriptase and colored dye are in the same container or separate containers suitable for combining together in a cDNA synthesis mix, wherein the colored dye is at a concentration that is in the range of 0.007%-1% in the kit. 
     
     
         8 . The kit according to  claim 7 , further comprising at least one component selected from the group consisting of buffer, glycerol or equivalent, dNTPs, nucleic acid primers, an RNAse inhibitor and DNA adaptors. 
     
     
         9 . A method comprising:
 combining an aliquot of the reverse transcriptase mixture of according to  claim 1  with reagents for cDNA synthesis, but not template single strand nucleic acid, to produce a cDNA synthesis mix; and   confirming that the reverse transcriptase mixture has been added to reagents by: (i) visually observing the color of the cDNA synthesis mix, and/or (ii) visually detecting a colored bead in the cDNA synthesis mix, such as wherein the visible colored bead has a greater density than the buffer in the cDNA synthesis mix.   
     
     
         10 . A method, comprising:
 (a) combining an aliquot of the reverse transcriptase mixture of  claim 6 , or an aliquot of the cDNA synthesis mix defined in  claim 9 , with a template single strand nucleic acid to produce a cDNA reaction mix; and   (b) incubating the cDNA reaction mix to produce a cDNA product.   
     
     
         11 . The method according to  claim 10 , wherein, after (b), the method comprises: removing a specific volume of the cDNA reaction mix containing the dye and the cDNA product and adding the specific volume of the cDNA reaction mix containing the dye and the cDNA product to a receiving mix. 
     
     
         12 . The method according to  claim 11 , wherein the receiving mix does not contain colored dye prior to the addition of the primary mix. 
     
     
         13 . The method according to  claim 11 , wherein the method further comprises:
 (i) visually observing the specific volume of the cDNA reaction mix removed from the cDNA reaction mix for adding to the receiving mix, such as by a series of micropipette tips; and/or   (ii) confirming that the specific volume of the cDNA reaction mix has been added to the receiving mix by observing visually the colored dye in the receiving mix; and/or by visually detecting a colored bead at or towards the bottom of a reaction tube containing the receiving mix, such as wherein the visible colored bead has a greater density than the buffer in the receiving mix.   
     
     
         14 . The method according to  claim 11 , wherein the receiving mix comprises at least PCR primers, dNTPs, and Taq polymerase; and wherein the addition of cDNA reaction mix containing the cDNA product to the receiving mix produces a PCR reaction mix, and the method further comprises incubating the PCR reaction mix to produce a PCR product. 
     
     
         15 . The method of  claim 14 , wherein the colored dye in the PCR reaction mixture is at a concentration of less than 0.007%. 
     
     
         16 . The method of  claim 14 , wherein the PCR reaction is a qPCR reaction.