The Countess™ II or Countess™ II FL instrument screen is dark on one side but brighter on the other. How can I fix this?

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Answer

Uneven illumination on the screen may be fixed by resetting the light cube tray. First, make sure you have the most recent version of the Countess™ II or Countess™ II FL software installed on the instrument (it is available here). If the software is updated, the light cube tray should readjust each time the instrument is power cycled. You can also select the “change light cubes” option under the settings menu, allow the light cubes to reset, and then turn the instrument off and back on. This process may help with uneven screen illumination even if you do not have light cubes present or if you have the Countess™ II instrument (without the fluorescence option).

Answer Id: E12359

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I don't want to stock twenty different resins for each amino acid. Can I buy a peptide synthesis resin without an amino acid and attach it myself?

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Answer

Yes. Resins for making peptide amides have no amino acid, nor do they need one. They have an Fmoc-protected amine.

Answer Id: E1235

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Is there a difference in how transfection efficiency is measured with the Neon ™ System versus the Nucleofector™ Device?

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The Neon™ system measures the transfection efficiency by the percentage of GFP-positive cells among all cells which include live and dead cells. In contrast, the Nucleofector™ Device measures the transfection efficiency by the percentage of GFP-positive cells among only the live cells.

Answer Id: E5490

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I’m trying to clone a gene that has multiple repeated sequences into my pCR™2.1-TOPO™ vector, followed by transformation into TOP10 cells. My clones contain random rearrangements and deletions. What can I do?

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Answer

With any strain, the first thing to try would be to lower the growth temperature of the culture to 40 degrees C or even lower (room temperature). Slower growth will generally allow E. coli to tolerate difficult sequences better. If reducing the growth temperature doesn’t help, you may want to consider using a competent cell strain such as Stbl2™ or Stbl4™ cells, which have been shown to accommodate this type of sequence better than other strains in the same conditions.

Answer Id: E6729

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What are the sequencing performance specifications for the different array lengths and polymer types for the Applied Biosystems™ 3130 Series Systems?

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On the 36 cm array:

- The UltraSeq36_POP7 run module has a run time of 35 minutes and an average read of 500 bases with a QV >20.

- The RapidSeq 36_POP7 run module has a run time of 60 minutes and an average read length of 600 bases with a QV >20.

- The UltraSeq36_POP4 run module has a run time of 40 minutes and an average read length of 400 bases with a QV >20.

- The RapidSeq36_POP6 run module has a run time of 60 minutes and an average read length of 500 bases with a QV >20.

On the 50 cm array:

- The FastSeq50 _POP7 run module has a run time of 60 minutes and an average read length of 700 bases with a QV >20.

- The StdSeq50 _POP7 run module has a run time of 120 minutes and an average read length of 850 bases with a QV >20.

- The StdSeq50 _POP4 run module has a run time of 100 minutes and an average read length of 600 bases with a QV >20.

- The StdSeq50 _POP6 run module has a run time of 150 minutes and an average read length of 600 bases with a QV >20.

On the 80 cm array:

- The LongSeq80 _POP7 run module has a run time of 170 minutes and an average read length of 950 bases with a QV >20.

- The LongSeq80 _POP4 run module has a run time of 210 minutes and an average read length of 700 bases with a QV >20.

Answer Id: E2386

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With the Countess™ II and Countess™ II FL instruments, the autofocus does not seem to focus on the cells very well. What can I do?

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Answer

First, make sure you have the most recent version of the Countess™ II or Countess™ II FL software installed on the instrument (it is available here). You may need to manually adjust the focus and set the nominal focus so that the autofocus will have a set point to focus on the cells. Once cells are stained 1:1 in 0.4% trypan blue and placed in the slide, you may want to let the cells settle for around 30 seconds. Then place the slide in the instrument and after you let the machine autofocus, you may need to refine the brightfield focus with the manual focus. Ideally the live cells should be slightly under focused, where the centers are bright compared to the dead cells that are dark throughout. You may want to zoom in so that you can better see the cells, then manually adjust the focus using the focus slider bar before you hit capture. Once the cells are focused, you can tap the blue “set” box to set the nominal focus; setting the nominal focus will improve autofocus consistency with future slides.
Make sure there are no bubbles or debris visible on the screen that could interfere with the autofocus and make it more difficult to get the sample in the correct focal plane. You can find some more guidelines for focusing starting on page 20 of the manual.

Answer Id: E12360

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How are the Neon™ Kit cell viability and transfection efficiency determined by your R&D team?

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Answer

Cells were analyzed for viability and transfection efficiencies 24 hours or 48 hours post-electroporation using a Guava PCA-96 Cell Analysis System. LIVE/DEAD™ cell viability assay populations were calculated by propidium iodide staining (1:2000). The percent of transfected cells was calculated by dividing the number of GFP positive cells by the total population and recorded as transfection efficiencies. The percent of dead cells was calculated by dividing the number of PI-stained cells by the total population

Answer Id: E5491

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My ZOOM™ Dual Power Supply stopped running with an alarm and the screen displays shows “OVER VOLTAGE”. What is the problem?

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Answer

This indicates that the circuit is interrupted. Please check the following:

- Verify that the running buffer is correct.
- Verify that all cables are attached correctly.
- Turn the Power switch off and on again; restart the application.
- If you cannot restart the instrument, turn off the power, disconnect the power cord from the outlet and contact Technical Support at techsupport@thermofisher.com.

Answer Id: E11053

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I’m trying to transform large plasmid, 40 kb in size. What strain should I use?

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Answer

While there is no specific strain that works better with large plasmids, it is important to focus on transformation efficiency. For larger plasmids, chemically competent cells with highest efficiency are suggested, such as OmniMAX™ 2, TOP10, etc. We would recommend using an electrocompetent cell strain with plasmids larger than 20 kb for best efficiency.

Answer Id: E6730

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When using SYBR™ Green dye chemistry on an Applied Biosystems™ 7900HT Fast Real-Time PCR System, is there an option to set the quencher to reflect the fact that there is no TaqMan™ probe being used in the reaction?

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Answer

Yes, you can set this as an option. On the Detector Manager for the Applied Biosystems™ 7900HT Fast Real-Time PCR System, simply set the Quencher to "Non Fluorescent".

Answer Id: E2390

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Can the direct lysis approach to DNA/RNA analysis be automated?

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Answer

Yes, Cells-to-CT™ kits are great for automation, including the screening of siRNAs or small molecules.

Answer Id: E7111

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Can T7, T3, SP6, and M13 primers be used in PCR?

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Answer

Yes, T7, T3, SP6, and M13 primers can be used in PCR. These primers may be used together or in conjunction with gene-specific primers for PCR of any of the Invitrogen™ vectors that contain these primer binding sites. However, note that optimization of annealing temperature may be needed as these primer sequences are slightly shorter than average PCR primers.

Answer Id: E3628

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Will washing in water affect the biotin-streptavidin bond to Dynabeads™ Streptavidin magnetic beads?

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Answer

After biotinylated DNA has been bound to Dynabeads™ Streptavidin magnetic beads, the complex can be washed in water without influencing the streptavidin or the binding.

Answer Id: E6177

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With the Countess™ II and Countess™ II FL instruments, images are very bright and washed out, or the fluorescence is extremely bright and bleeding through into other filters. How can I adjust this?

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Answer

Decrease the brightfield and fluorescence light intensity before counting the cells.

Answer Id: E12361

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How is Neon™ System transfection efficiency measured?

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Answer

We normally analyze transfection efficiency in living cells by FACS. We first exclude cellular debris by gating for the "normal" population (regarding size and granularity) in the forward-sidescatter. From this gated population we determine dying cells by propidium iodide staining and exclude them from analysis by setting another gate. For example, in one experiment we transfected Human primary CD4+T-cells with 2250V, 20ms, 1 and 0.5 ug EGFP in primary blood buffer using the Neon™ System. 24 hrs post electroporation, cells were analyzed by flow cytometry. CD4+T-cell were gated according to forward/side scatter, and dead cells were excluded by propidium iodide staining and gating. GFP gene expression of T cells was measured after electroporation with plasmid DNA, and we found 32.74% transfection efficiency and 75% viability. Please see our "Learn More about Neon™ Transfection System" pages (search this term from our home page) for more data and information.

Answer Id: E5492

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