RT-PCR Obtain the internal region of cDNA 1. Mix crude RNA or poly(A) RNA, cDNA primer (one of the amplification primers in 7.) 2. Heat at 65 0 C for 3-15 min. 3. Cool on ice 4. Add RT and RT buffer, 42 0 C (37 0 C to 55 0 C, depending on the composition of primer and RNA) for 1 hr. 5. Dilute and phenol extraction 6. Ethanol precipitation 7. Mix cDNA, amplification primers, dNTPs, 94 0 C for 2 min. 8. Add Taq polymerase, run PCR. 9. Analyze PCR products on agarose or nondenaturing PAGE.

To obtain full-length cDNA. (BD Clontech) 3 ’ -RACE, and 5 ’ -RACE amplification kits to obtain upstream and downstream cDNA, and clone.

Chapter 23: Discovery and analysis of different genes or differentially expressed genes

With two bacteria, (BD-clontech) PCR-select Genomic substraction kit 1.Construct gene library with genes markedly different in the two bacteria (eg. presence or absence of pathogenicity islands). 2. Then use genomic DNA probes (radiolabelling by random-priming) of both bacteria to check the colonies of the library.

To generate differentially expressed library (BD-clontech) PCR-Select cDNA substraction kit 1. Prepare tester cDNA and driver cDNA (in excess) 2. Add adaptors 3. Two rounds of hybridization 4. PCR

To find the differentially expressed genes from the differentially expressed library (BD-clontech) PCR-Select Differential screening kit Colonies hybridized with tester cDNA probe, driver cDNA probe, substracted cDNA probe, and non- substracted probe. (Colonies with +, －,+, － are the differentially expressed genes; Colonies with, －, －, +, － are very likely the differentially expressed genes)

(BD-clontech) Delta differential display kit. Using sets of T primers and ten arbitrary primers that found by computer analysis of the coding regions of more than 200 mRNAs 1. Prepare first-strand ss cDNA of two samples 2. PCR with α32P-dATP, 3 low-stringency cycles and high-stringency cycles. 3. 5% non-denaturing PAGE and autoradiography 4. Clone the fragments different with the two samples.

AFLP (amplification fragment length polymorphisms) (unit 23.5) 1. Genomic DNAs or cDNAs of many samples. 2. Two rounds of PCR 3. Denaturing sequencing gel 4. Eluting the ssDNA fragment 5. Polymerization by klenow 6. Clone

From BRL

Chapter 19: Analysis of protein interaction

In vivo two-hybrid systems

(BD) Matchmaker yeast 2-hybrid system 3 - Use the binding and activation domains of yeast Gal4 transcriptional factor (B42 is an artificial Gal4 activation domain). - NLS (nuclear localization sequence) is added to both domains. - Reporter gene can be on plasmid or chromosome. - Binding sequence upstream of reporter gene: GAL4uas Other systems using SOS box (or LexAop) as binding sequence and LexA protein as BD, to avoid the endogenous GAL4uas and Gal4 protein in yeast.

(BD) Matchmaker mammalian 2-hybrid kit Use mammalian vectors pM: contains p53 as BD pVP16: contains vp16 as AD pGSCAT : reporter plasmid, protein interaction induce CAT expression in cells. pGSSEAP : reporter plasmid, interaction induce SEAP expression in culture medium. Co-transfect cells with pM, pVP16, and pGSCAT / pGSSEAP, incubate for 48 hrs, and assay for CAT or SEAP activity.

(Stratagene) BacterioMatch 2-hybrid system Clone bait gene to the C-terminus of cI in pBT, and the interacting target gene to the N- terminus of the α subunit of RNA polymerase in pTRG. Reporter genes in an operon for histidine prototrophy and streptomycin resistance. Many pre-made mammalian cDNA libraries in pTRG are available.

(Stratagene) Cytotrap 2-hybrid system For detection of protein-protein interactions occurring on the cell membrane. - pMyr vector: contains BD which will be myristilated and binds (inserts) to cell membrane. - pSos vector: contain human homolog of CDC25 (hSos), encoding GTP exchanging factor (GEF), as AD. (GEF will bind Ras and activate Ras signal pathway to allow yeast cells to grow.) - cdc25H yeast: a cdc25 ts yeast mutant. Cells grow normally at 25 0 C, but cannot grow at 37 0 C. Protein interaction will allow the cdc25H yeast to grow at 37 0 C.

Affinity purification of proteins binding to GST fusion proteins (GST pulldown assay) 1. Prepare E. coli cell extract (competitor, to remove background). 2. Mix 35S-radiolabelled test protein and E. coli cell extract. Incubate 15 min on ice. 3. Add GST or GST-protein bound to agarose beads to cell extract in an eppendorf tube, Mix. 4. Add 3. to 2. Incubate 1-2 hr at 40C. 5. Centrifuge to remove supernatant. Wash beads with binding buffer. 6. Add SDS sample buffer. Boil for 5 min, SDS-PAGE and autoradiography. Preparation of E. coli cell extract by sonication.

Surface Plasmon Resonance (SPR) using BIAcore (detection) 1. Assemble NTA chips in BIAcore. 2. Inject 10ul of 1 mM NaOH 3. Inject His-tag protein 4. Inject target protein 5. Measure dissociation and association rates 6. Use 200 mM immidazole to wash the His-tag protein and the His-tag protein complex. Can use amine-coupling kit (BIAcore) to coating target proteins on the nude chip.

Detection of protein-protein interaction by co-precipitation. 1. Prepare whole cell extract 2. Incubate with antibody against protein 1 3. Incubate with protein-A sepharose 4. Centrifuge to collect beads 5. Wash pellet 6. Dissociate proteins from protein A-sepharose by adding SDS sample buffer and boiling. 7. Supernatant for SDS-PAGE 8. Immunoblot with antibody against protein Re-probe with antibody against protein 2.

Identification of protein interactions by far western analysis. Protein samples immobilized on PVDF membranes, and probed with A)an 35 S-labelled non-antibody protein, or B)an unlabelled protein probe, and further detected with a probe-specific antibody.