Biomedical Engineering Reference
In-Depth Information
Table 1
Primers used for TPCR cloning of IFN
α
8 gene into pET28-TevH and subsequent analyses
Recipient
plasmid
Gene/task
Primer name (length)
Primer sequence (5′
⇒
3 ′)
IFN
α
8
pET28-TevH
TPIFN8_30olF
(53 bases)
TCCGCGGGTGAAAACCTGT
ACTTCCAGGGT
TGTGATC
TGCCTCAGACTCACAG
TPIFN8_30olR
(57 bases)
GTGGTGGTGCTCGAGTGCG
GCCGCAAGCTT
CTATTCC
TTACTCTTCAATCTTTTTTG
Colony PCR
-
T7 (23 bases)
ATTAATACGACTCACTATAGGGG
PetRev (22 bases)
ATGCTAGTTATTGCTCAGCGGT
IFN
8 sequences are underlined (marked in red in Fig.
2b
). The T7 and PetRev primers are used for colony PCR
(
see
Subheading
3.3
) and sequencing analyses
α
9. High effi ciency
E. coli
DH5
α
competent cells for heat shock
transformation (
see
Note 3
).
10. Sterile spreaders.
11. Lysogeny Broth (LB) agar plates supplemented with Kanamycin
(30
g/mL).
12. 37 °C incubator.
μ
1. Synthetic primers for colony PCR and sequencing analyses (
see
Table
1
).
2. Agarose gel electrophoresis apparatus.
3. Agarose I (Amresco, Solon, OH; Catalog # 0710) or equivalent.
4. Ethidium bromide dropper bottle, 0.625 mg/mL stock
(Amresco, Solon, OH; Catalog # E406) or equivalent.
5. Tris/acetic-acid/EDTA (TAE) buffer, 50× stock (Bio-Rad
Laboratories, Hercules, CA; Catalog # 161-0743) or equivalent.
6. Microwave oven.
7. Gel-Doc 2000 visualization system (Bio-Rad Laboratories,
Hercules, CA; Catalog # 170-8126) or similar.
8. 50 mL round conical centrifuge tubes (BD Falcon, Franklin
Lakes, NJ; Catalog # 352077) or equivalent.
9. Lysogeny Broth (LB) liquid medium supplemented with
Kanamycin (30
2.3.2
Analysis
μ
g/mL).
