Viral Vectors to Study Synaptic Function - Viral Vector Approaches in Neurobiology and Brain Diseases - page 113

5. Restriction and ligation enzymes and buffers (e.g., XbaI, SphI,
T4 ligase)
6. DNA amplifi cation and purifi cation kits (e.g., QIAprep Spin
Miniprep kit and QIAquick PCR purifi cation kit or QIAquick
Gel Extraction kit, QIAGEN)
7. Electro-competent or chemically competent Escherichia coli
8. Sequencing primers (the pSINREP5 primer is provided in
Fig. 1b , which permits sequencing downstream of the multiple
cloning site of the sindbis virus-based vectors)

1. Level 2 biosafety tissue culture room including tissue culture
hood and incubator
2. Restriction enzyme for linearization (XhoI or PacI or NotI)
3. Helper DH-BB(tRNA/TE12) DNA [ 20 ]
4. Cloned recombinant sindbis virus DNA (Sect. 3.1.1 )
5. DNA amplifi cation kit (e.g., QIAfi lter Plasmid Maxi kit)
6. DNA purifi cation kit (e.g., QIAquick PCR purifi cation kit)
7. In vitro transcription kit (e.g., mMESSAGE mMACHINE ®
SP6 Kit, cat. no. AM1340, Ambion, Inc., USA)
8. Tissue culture plastic ware including 150-mm 2 plates and
T150 mm 2 fl asks with fi ltered caps
9. Phosphate buffer saline (PBS) with cations, i.e., MgCl 2 and
CaCl 2
10. RNAse-free PBS without cations
11. Trypsin-EDTA
12.

2.1.2 Production
and Concentration
of Sindbis Viruses

MEM medium with l-glutamine
13. Fetal bovine serum (FBS)
14. Bright fi eld microscope and cell counting instruments
15. Electroporator for mammalian cells (e.g., Biorad Gene Pulser
XCell)
16. Biorad electroporation cuvettes (0.4 cm)
17. Beckman ultracentrifuge
18. Beckman rotor SW40Ti (swinging buckets)

α

19. Beckman ultra-clear centrifuge tubes (cat. no. 344059)

2.2 In Vivo
Stereotaxic Injection
of Viruses

1. Rat or mouse stereotaxic frame (depending on species to be
used for the studies)
2. Microdrill
3. High accuracy pump (e.g., Harvard PHD2000 or KSD
legato 110)
4. 5

L injection syringe (e.g., SGE Analytical science cat. no.
5F-HP-0.63/0.47)

μ