Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V012837 | pPA-TagRFP-H2B | In stock, instant shipping |
Buy one, get one free! |
Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.
Basic Vector Information
pPA-TagRFP-H2B is a mammalian expression vector, which encoding the PA-TagRFP-H2B fusion protein. The vector can be used for fluorescent labeling of histone H2B in living cells.
- Vector Name:
- pPA-TagRFP-H2B
- Antibiotic Resistance:
- Kanamycin
- Length:
- 5089 bp
- Type:
- Fluorescent Protein Genes & Plasmids
- Replication origin:
- ori
- Source/Author:
- Evrogen
- Selection Marker:
- Neomycin (select with G418)
- Copy Number:
- High copy number
- Promoter:
- CMV
- Cloning Method:
- Enzyme digestion and ligation
- Fusion Tag:
- PA-TagRFP
- Growth Strain(s):
- Top10
- Growth Temperature:
- 37℃
pPA-TagRFP-H2B vector Vector Map
Plasmid Protocol
1. Centrifuge at 5,000×g for 5 min.
2. Carefully open the tube and add 20 μl of sterile water to dissolve the DNA.
3. Close the tube and incubate for 10 minutes at room temperature.
4. Briefly vortex the tube and then do a quick spin to concentrate the liquid at the bottom. Speed is less than 5000×g.
5. Store the plasmid at -20 ℃.
6. The concentration of plasmid re-measurement sometimes differs from the nominal value, which may be due to the position of the lyophilized plasmid in the tube, the efficiency of the re-dissolution, the measurement bias, and adsorption on the wall of the tube, therefore, it is recommended to transform and extract the plasmid before using it
General Plasmid Transform Protocol
1. Take one 100μl of the competent cells and thaw it on ice for 10min, add 2μl of plasmid, then ice bath for 30min, then heat-shock it at 42℃ for 60s, do not stir, and then ice bath for 2min.
2. Add 900μl of LB liquid medium without antibiotics, and incubate at 37℃ for 45min (30℃ for 1-1.5 hours) with 180rpm shaking.
3. Centrifuge at 6000rpm for 5min, leave only 100μl of supernatant to resuspend the bacterial precipitate and spread it onto the target plasmid-resistant LB plate.
4. Invert the plate and incubate at 37℃ for 14h, or at 30℃ for 20h.
5. Pick a single colony into LB liquid medium, add the corresponding antibiotics, incubate at 220rpm for 14h, and extract the plasmid according to the experimental needs and the instructions of the plasmid extraction kit.
References
- Nijenhuis W, Vallardi G, Teixeira A, Kops GJ, Saurin AT. Negative feedback at kinetochores underlies a responsive spindle checkpoint signal. Nat Cell Biol. 2014 Dec;16(12):1257-64.
pPA-TagRFP-H2B vector Sequence
LOCUS 40924_34037 5089 bp DNA circular SYN 01-MAR-2024
DEFINITION synthetic circular DNA.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 5089)
AUTHORS .
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..5089
/mol_type="other DNA"
/organism="synthetic DNA construct"
enhancer 61..364
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 365..568
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
CDS 657..1034
/label=H2B
/note="human histone H2B"
CDS 1053..1751
/label=PA-TagRFP
/note="photoactivatable variant of the monomeric red
fluorescent protein TagRFP (Subach et al., 2010)"
polyA_signal 1877..1998
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
rep_origin complement(2005..2460)
/direction=LEFT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 2487..2591
/label=AmpR promoter
promoter 2593..2950
/label=SV40 promoter
/note="SV40 enhancer and early promoter"
CDS 2985..3776
/label=NeoR/KanR
/note="aminoglycoside phosphotransferase"
polyA_signal 4011..4058
/label=HSV TK poly(A) signal
/note="herpes simplex virus thymidine kinase
polyadenylation signal (Cole and Stacy, 1985)"
rep_origin 4387..4975
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"