Part A - Production of PCR-ready, linkered, ds cDNA

1.	Set up the following reverse transcription reaction. 4.5 µl RNA/H 20 1 µl oligo d(T)12-18 (total of 500 ng) Note: Total RNA, 10 - 100 ng, can be used in this protocol.
2.	Heat this mixture to 65°C for 10 minutes.
3.	Place directly at 42-45°C for 5 minutes.
4.	Add the following reagents that have been pre-mixed (4.5 µl volume) and pre-heated to 42-45°C for 2 minutes: 2 µl 5X first strand buffer 1 µl 100 mM DTT 0.5 µl 10 mM dNTP 1 µl Superscript II RT »Total reaction volume = 10 µl
5.	Incubate this reaction at 42-45°C for 30 additional minutes.
6.	Place on ice to stop the reaction.
7.	Set up the second strand replacement reaction on ice by adding the reagents listed below in the order in which they appear. 10 µl RT reaction 101 µl H2O 30 µl 5X second strand buffer 3 µl 10 mM dNTPs 1 µl E. Coli DNA Ligase 4 µl E. Coli DNA Pol I 1 µl E. Coli Rnase H »Total reaction volume = 150 µl
8.	Incubate at 16°C for 2 hours.
9.	Add 2 µl of T4 DNA Polymerase.
10.	Continue incubating at 16°C for an additional 10 minutes.
11.	Place the tube on ice to stop the reaction.
12.	Add 150 µl phenol:chloroform:isoamyl (24:25:1) and vortex completely.
13.	Spin at 14,000 rpm for 10 minutes at room temperature.
14.	Transfer the upper phase to a new microfuge tube and add 150 µl chloroform:isoamyl.
15.	Centrifuge at 14,000 rpm for 5 minutes at room temperature and transfer the upper phase to a fresh microfuge tube.
16.	Add 1 µl glycogen carrier and 150 µl isopropanol.
17.	Keep at -20°C for at least 20 minutes.
18.	Precipitate by centrifuging at 14,000 rpm for 20 minutes at 4°C.
19.	Wash pellet with 70% ethanol, air dry pellet, and resuspend cDNA in 27 µl of sterile H 2O.
20.	Set up the adaptor ligation reaction on ice according to the following: 27 µl cDNA suspension 10 µl 5X adaptor buffer 1 µl EcoRI adaptors (1µg/ml) 7 µl 0.1M DTT 5 µl T4 DNA Ligase »Total reaction volume = 50 µl
21.	Incubate the reaction at 16°C for a minimum of 16 hours. It is easiest to allow this step to proceed overnight.
22.	Purify the adaptor-ligated cDNA from unligated adaptors and small molecular weight cDNA by agarose gel electrophoresis. Pour a 1.0% low melting point agarose gel. Load the ligation reaction in the gel and run at 30-60 volts long enough to easily resolve 300 to 400 bp fragments by comparison to 100 bp molecular weight marker. Cut out the cDNA that resides between the 400 bp and 2,000 bp markers. Place the gel slice in a 1.7 ml microfuge tube.
23.	Melt the gel slice at 65°C just long enough to melt the gel mixture (usually 15-20 minutes).
24.	Add an appropriate amount of 10X beta agarase buffer, 20 µl beta-agarase enzyme, and incubate overnight at 37°C.
25.	Extract the ligated cDNA as follows: Add equal volume phenol:chloroform:isoamyl alcohol (24:25:1). Vortex thoroughly. Centrifuge at RT for 10 minutes at 14,000 rpm. Remove the upper layer and place in a fresh microfuge tube. Add equal volume chloroform, vortex. Centrifuge at RT for 5 minutes at 14,000 rpm. Remove the upper layer and place in a fresh microfuge tube followed by addition of equal volume isopropyl alcohol and 1 µl of glycogen (20 µg/ml). Place tube at -20°C for at least 20 minutes. Precipitate by centrifuging at 14,000 rpm for 20 minutes at 4°C. Wash pellet once with 70% ethanol and air dry. Resuspend in 20 µl sterile H2O.
26.	This cDNA is now ready for PCR.

Part B - PCR Amplification of ds cDNA

1.	Perform the following PCR reaction (for amplification and subcloning purposes): 20 µl cDNA 63.5 µl H2O 10 µl 10X PCR buffer 2.5 µl 10 mM dNTPs 2 µl 50 µm LINK-CUA primer 1 µl Taq polymerase 1 µl Perfect Match »Total reaction volume = 100 µl Notes: a) Taq polymerase can be acquired from one of many commercial suppliers. It is important to use the 10X PCR buffer that is included with the purchased Taq to get maximum amplification efficiency. b) The LINK-CUA primer is designed specifically for the EcoRI adaptor ligated to the ends of all ds cDNA molecules. The sequence is as follows: 5' CUACUACUACUAAATTCGCGGCCGCGTCGAC 3' c) The triplet repeat CUA must be present on this primer for the purposes of UDG cloning of PCR product as discussed later. d) Always set up and amplify a negative control in parallel. A negative control consists of the same reaction as described above but lacking template. Thus instead of the 2 µl of cDNA simply add 2 µl of H2O in its place.
2.	Perform the PCR reaction in a thermalcycler according to the following cycling conditions: #Cycles Temperature °C Time 1 94 3 min 20 94 68 72 15 sec 15 sec 3 min 1 72 5 min
3.	Hold at 4°C.
4.	Analyze 10 µl of both the cDNA PCR reaction as well as the negative control by 1.2% agarose gel electrophoresis. Notes: a) A successful library will be reflected by a faint homogeneous smear of product ranging in size from 400 bp to 1,500 bp as compared to 100 bp molecular weight marker. b) The average cDNA size should be roughly 500 bp. c) The negative control should give no PCR product for a library to be considered successful.
5.	Purify the final 20-cycle PCR product as follows: Add 100 µl phenol:chloroform:isoamyl alcohol (24:25:1). Vortex thoroughly. Centrifuge at room temperature for 10 minutes at 14,000 rpm. Remove the upper layer and place in a fresh microfuge tube. Add equal volume chloroform. Vortex, and centrifuge 5 minutes at 14,000 rpm. Remove the upper layer and place in a fresh microfuge tube. Add 100 µl isopropyl alcohol and 1 µl of glycogen (20 µg/ml). Place tube at -20°C for at least 20 minutes. Centrifuging at 14,000 rpm for 20 minutes at 4°C. Wash pellet once with 70% ethanol, air dry, and resuspend in 10 µl sterile H2O.
6.	The amplified library is ready to clone into the plasmid vector pAMP10 using the UDG subcloning protocol.

Part C - Subcloning of cDNA Library

All reagents necessary to carry out UDG cloning are taken from the CloneAmp cloning kit from Life Technologies (Cat.# 18552-018).