FastDigest® PfoI
Detailní popis
Thermo Scientific FastDigest enzymes are an advanced line of fast restriction enzymes that are all 100% active in the universal FastDigest and FastDigest Green reaction buffers.
The universal buffer allows rapid single-, double- or multiple DNA digestion within 5-15 minutes eliminating any need for buffer change or subsequent DNA clean-up steps. DNA modifying enzymes, such as Klenow Fragment, T4 DNA Ligase, alkaline phosphatases and T4 DNA Polymerase all have 100% activity in FastDigest Buffer. Therefore, enzymes for downstream applications can be directly added to the FastDigest reaction mix.
For additional convenience FastDigest Green Buffer includes a density reagent and two tracking dyes for direct loading of digestion reaction products on gels.
Short incubation times and optimal composition of the universal FastDigest Buffer eliminate star activity effects.
Features
- 100% activity of all FastDigest enzymes in the universal buffer
- 100% buffer compatibility with downstream applications
- Complete digestion in 5-15 minutes
- Direct loading on gels
- No star activity
- 176 FastDigest enzymes available
Applications
- Molecular cloning
- Restriction site mapping
- Genotyping
- Southern Blot
- Restriction fragment length polymorphism (RFLP)
- SNP analysis
Note
The FastDigest Green Buffer offers the same high performance in DNA digestion and downstream applications as the colorless FastDigest Buffer. For applications that require product analysis by fluorescence excitation (e.g. concentration measurements in UV light) the colorless FastDigest Buffer is recommended.
For methylation sensitivity refer to product specifications.
Reaction conditions
| Reaction temperature | Digestion time with 1 µL of FastDigest enzyme, min | bp from end of DNA required for complete digestion | Thermal inactivation | Incubation time without star activity, hours | |||
|---|---|---|---|---|---|---|---|
| Lambda, 1 µg/20 µL | Plasmid DNA, 1 µg/20 µL | PCR product, ~0.2 µg/30 µL | Genomic DNA, 1 µg/10 µL | ||||
| 37°C | 5 | 5 | 5 | 5 | 3 | 65°C, 5 min | 16 |
Methylation Effects
- Dam: may overlap - cleavage impaired.
- Dcm: may overlap - blocked.
- CpG: may overlap - blocked.
- EcoKI: never overlaps - no effect.
- EcoBI: never overlaps - no effect.
| Methylation type | Sequence | Cleavage effect |
|---|---|---|
Dam(GATC) |
5'...TCCNGGm6A TC...3'3'...AGGNCC Tm6AG...5' |
Impaired |
Dcm(CCWGG) |
5'...TCm5CA GGA...3'3'...AG GTm5CCT...5' |
Blocked |
| CpG | 5'...TCm5C GGGA...3'3'...AG Gm5CCCT...5' |
Blocked |
Number of recognition sites in DNA molecules
| Lambda | ΦX174 | M13mp18/19 | pBR322 | puc18/19 | pUC57 |
|---|---|---|---|---|---|
| 15 | 0 | 0 | 1 | 1 | 1 |
| pTZ19R/U | pTZ57R | pBluescriptIIKS(±) | pBluescriptIISK(±) | pACYC177 | pACYC184 |
| 0 | 0 | 0 | 0 | 1 | 1 |
New sites generated by ligation
Newly generated recognition sites resulting from fill-in of 5'-overhang and self-ligation
| Recognition Sequence | Newly generated sequence after reaction | Enzymes that cleave the newly generated sequence |
|---|---|---|
T^CCNGGA |
TCCNGGCCNGGA |
|
T^CCAGGA |
TCCAGGCCAGGA |
|
T^CCCGGA |
TCCCGGCCCGGA |
|
T^CCGGGA |
TCCGGGCCGGGA |
|
T^CCTGGA |
TCCTGGCCTGGA |
|
Obsah balení
20 rxns
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