STEWART LABORATORY

 

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cDNA SYNTHESIS

 

RNA ISOLATION

 

             Grow cells to 50-60% confluency (log phase of growth)

             Isolate RNA using RNA-STAT 60 (CS-110 from Tel-Test, Inc. 1-800-631-0600)

 

 

POLY A SELECTION

 

             Isolate polyA RNA by using Qiagen Oligotex Kit ( 70042)

 

 

CDNA SYNTHESIS

 

                                                      1.            Use kit from Life Technologies (Superscript Choice System for cDNA synthesis 18090-019). The kit is actually good for 5 reactions

                                                      2.            Synthesize using 5 μg of Poly A RNA (ppt 5 μg the night before / spin the day of synthesis / re-suspend according to protocol from kit)

                                                      3.            Do not use radioactivity to measure your synthesis

                                                      4.            For first strand synthesis, I use 1 μL of water instead of dCTP

                                                      5.            For second strand synthesis, I use 91 μL of water instead of 93 because I did not take any of the first strand synthesis out for anaylsis

                                                      6.            By the end of step 4 on second strand synthesis, I have a total of 162 μL. I phenol chloroform extract the whole 162 μL and take 150 μL for ethanol ppt.
(75 μL of 7.5 M ammonium acetate with 536 μL of ethanol)

 

 

ADAPTOR LIGATION

 

                                                      1.            Re-suspend pellet from ethanol ppt with 23 μL of water

 

                                                      2.            Use fresh Ligase from New England Biolabs (202S)

 

                                                      3.            Ligase mix:

 

         10x Ligase Buffer : 3 μL

         T4 DNA ligase: 2 L

         cDNA: 23 L

         BstX1/EcoR1: 2 L (Invitrogen N418-18, dissolved to 1 g/L conc)

 

 

 

                                                      4.            Incubate at room temperature for 4 hours and move to 16C for overnight incubation

 

 

PURIFICATION

 

                                                      1.            Pour low-melt agarose gel (0.8%) (SeaPlague GTG Agarose from American Bioanalytical AB197). Choose a comb size that fits 40 μL in each well

 

                                                      2.            Heat inactivate ligation mixture and run on gel at 80V for 1-1.5 hour. Enough to separate out 1-3 kb range

 

                                                      3.            Take a picture of gel using fluorescent ruler with low UV illuminator

 

                                                      4.            Figure out measurements to take 3 fractions: 500bp-1kb, 1kb - 3kb, and 3kb-10kb

 

                                                      5.            Purify DNA away from agarose by agarse (Boeringer Mannheim, 1-417-223)

 

                                                      6.            Use 2x the amount of enzyme required

 

                                                      7.            After incubation, go directly to phenol:chloroform

 

                                                      8.            Ethanol ppt DNA overnight, using mussel glycogen as carrier

 

 

VECTOR LIGATION

 

                                                      1.            Re-supend cDNA in 21 μL

 

                                                      2.            Take 1/3 of cDNA 7 μL for ligation reaction

 

                                                      3.            50 ul Ligation mixture:

 

         7 μL of cDNA

         75-100 ng of retroviral vector

         5 μL of 10X Ligase Buffer

         3 μL of Ligase

 

 

 

                                                      4.            Incubate at 16C overnight

 

                                                      5.            Heat inactivate

 

                                                      6.            Phenol chloroform extract and ethanol ppt (overnight) with mussel glycogen

 

                                                      7.            Re-suspend in 20 μL of water

 

                                                      8.            Electroporate with ~7 μl of 3-10kb ligation, 3-4 μL of 1-3 kb ligation DH10B (Life Technologies, 18297-010) for about 1 million colonies

 

                                                      9.            Plate on sixteen 500 cm2 plates from Corning (431110 also available from VWR) per each electroporation. Incubate plates overnight

 

                                                 10.            Scrape plates. Four plates per Qiagen mega-column