| 1. General |
|
1.1 |
|
What cell lines and systems can be used with RheoSwitch? |
|
1.2 |
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Can RheoSwitch be used in mice? |
| 2. RSL1 Ligand |
|
2.1 |
|
Is RSL1 stable at room temperature? |
|
2.2 |
|
Can RSL1 be diluted in media? |
|
2.3 |
|
What is the maximum amount of RSL1 I can add to my cell culture? |
|
2.4 |
|
What is the maximum volume of RSL1 that I can add to my cell culture? |
| 3. pNEBR-X1 |
|
3.1 |
|
Does the pNEBR-X1 expression plasmid have an ATG? |
|
3.2 |
|
Does NEB sell a primer I can use to sequence from the 3' end? |
|
3.3 |
|
I have cloned my gene into pNEBR-X1. Can I make a stable cell line with this
plasmid? |
| 4. pNEBR-R1 |
|
4.1 |
|
How can I assay for expression of the RheoReceptor and RheoActivator genes? |
|
4.2 |
|
Can I make a stably transformed cell line using pNEBR-R1? |
|
4.3 |
|
What concentration of G418 should I use to construct a stable cell line with
pNEBR-R1? |
|
4.4 |
|
Once a stable cell line expressing with pNEBR-R1 is established, do I need
to continue culturing the cell line with G418? |
| 5. Gaussia Luciferase |
1. General |
|
| 1.1 What cell lines and systems can be used with RheoSwitch? |
| |
RheoSwitch is functional in a variety of mamalian cell lines and primary cell cultures. RheoSwitch is
functional in mice and NEB is exploring other in vivo systems as
well. Contact Technical Support, or e-mail
for more details.
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|
| 1.2 Can RheoSwitch be used in mice? |
| |
An earlier version of RheoSwitch that used RSL1 has been used successfully in mice. The RSL1 ligand
has no adverse affects in mice and good induction of a reporter gene is observed. Contact Technical Support,
or e-mail for more details.
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| |
| 2. RSL1 Ligand |
|
| 2.1 Is RSL1 stable at room temperature? |
|
While RSL1 dissolved in DMSO or in cell culture media is stable at room temperature for short periods
of time (days), the recommended storage conditions for RSL1 are -20°C in DMSO.
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|
| 2.2 Can RSL1 be diluted in media? |
|
RSL1 has poor solubility in aqueous solutions. However, it can be dissolved in media at 500 nM, the
recommended molarity for maximum induction. The media/RSL1 solution can then be stored at 4°C for a period
of weeks.
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|
| 2.3 What is the maximum amount of RSL1 I can add to my cell culture? |
|
NEB recommends a final RSL1 concentration of 500 nM for optimum expression of the cloned gene. While
higher amounts of RSL1 can be added to the cell culture, the increase in expression is then no longer linear.
RSL1 starts to loose solubility in culture media at approximately 1 mM final concentration.
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|
| 2.4 What is the maximum volume of RSL1 that I can add to my cell culture? |
|
DMSO may adversely affect cells in culture at high concentrations. We recommend a final DMSO concentration
in cell culture of less than 0.5%.
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| |
| 3. pNEBR-X1 |
|
| 3.1 Does the pNEBR-X1 expression plasmid have an ATG? |
|
pNEBR-X1 is designed to express your gene of interest as an unfused protein. The plasmid does not contain
an initiation codon nor 'Kozak' consensus sequences for optimal translation initiation. However, there
is an ATG as part of the ApaI site in the polylinker and this ATG can be used as an initiation codon.
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|
| 3.2 Does NEB sell a primer I can use to sequence from the 3' end? |
|
T7 Minimal Primer (19-mer) (NEB# S1272) can be used to obtain sequence from the 3' end of the MCS.
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|
| 3.3 I have cloned my gene into pNEBR-X1. Can I make a stable cell line with this plasmid? |
|
pNEBR-X1 does not have a selectable marker. However, stable transformants can be generated by co-transfecting
pNEBR-X1 with another plasmid that contains a selectable marker. Alternatively, your gene of interest can
be subcloned into pNEBR-X1Hygro (NEB# N8083), which carries the hygromycin resistance marker.
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| |
| 4. pNEBR-R1 |
|
| 4.1 How can I assay for expression of the RheoReceptor and RheoActivator genes? |
|
Transcription can be assayed by standard RT-PCR or qPCR protocols using primers designed specifically
to the two genes. Translation can be assayed by standard western protocols using sera to GAL4 (DBD) to
detect RheoReceptor and sera to VP16 activation domain to detect RheoActivator. Contact Technical Support,
or e-mail for more details.
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|
| 4.2 Can I make a stably transformed cell line using pNEBR-R1? |
|
pNEBR-R1 has a neomycin resistance marker, allowing selection of stable transformants using G418. Either
linear or supercoiled pNEBR-R1 plasmid may be used to generate stable lines. Care should be taken to avoid
linearizing pNEBR-R1 in such a way as to separate receptors from their promoters.
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|
| 4.3 What concentration of G418 should I use to construct a stable cell line with pNEBR-R1? |
|
Each mammalian cell line has different sensitivities to different antibiotics. The sensitivity can be
determined by performing a standard kill curve. The least amount of antibiotic that results in 100% lethality
over the course of a few days should be used for subsequent experiments.
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|
| 4.4 Once a stable cell line expressing with pNEBR-R1 is established, do I need to continue
culturing the cell line with G418? |
|
The stability of each cell line is different therefore, we recommend culturing cells in the presence
of the appropriate concentration of G418.
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| |
| 5. Gaussia Luciferase |
|
For questions about Gaussia Luciferase go to the FAQ page for Gaussia Luciferase
Assay Kit (NEB# E3300).
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