STEWART LABORATORY

cDNA SYNTHESIS

RNA ISOLATION

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

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

POLY A SELECTION

Ø 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 µL

§ cDNA: 23 µL

§ BstX1/EcoR1: 2 µL (Invitrogen N418-18, dissolved to 1 µg/µL conc)

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