Product FAQ

My Bolt™ Bis-Tris Plus gel is running backwards in the Bolt™ Mini Gel Tank but if I reverse the leads, it runs correctly. Can you please help me troubleshoot?

Answer

It is likely that the Bolt™ gel cassette was inserted backwards into the unit (large plate facing the front and the wells facing the back) even though this is pretty difficult to do. When the gel is inserted backwards, the current flows from the bottom of the gel to the top, resulting in the samples running in the opposite direction. Reversing of the leads will switch the direction of the gel run, however, this will cause the current to flow from the anode to the cathode. The cathode electrode is made of stainless steel with platinum coating, and the anode electrode is made of platinum wire. Flow of electrons from the anode to the cathode will result in rusting of the steel core. On the other hand, when the leads are connected properly, the electrons flow from the cathode to the anode and the recipient of the electrons is the platinum wire that does not rust. Note: When the gel cassette is inserted properly, the lettering (gel type, SKU and expiration date) printed on the cassette will be backwards (reads from right to left).

Answer Id: E11002

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Product FAQ

How can I determine protein concentration in buffers containing imidazole, pH 7.0?

Answer

We offer a Quant-iT™ Protein Assay Kit (Cat. No. Q33210), which is more sensitive than standard absorbance-based assays and can quantitate proteins from 0.25-5 μg. The signal is unaffected by many common contaminants, such as DTT, beta-mercaptoethanol, amino acids, and DNA. Imidazole at a final concentration below 1.25 mM is acceptable. Above that concentration, the imidazole begins to interfere with the assay. Please note, imidazole does absorb at 280 nm, and the absorbance varies with concentration. So to be perfectly accurate, each eluted fraction should be blanked against its elution buffer.

Answer Id: E12941

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Product FAQ

I am running a Bolt™ Bis-Tris Plus gel using the Bolt™ Mini Gel Tank and only one gel is running while the other isn’t. What is the issue?

Answer

Here are some likely reasons and remedies:

- Leaking of buffer from the inside of the tank to the outside. Leaking is most likely due to inappropriate removal of cassettes, which can lead to the gasket not sitting properly and loss of a good seal. We recommend pushing the gasket down with the thumb.
- Not covering the wells completely with the buffer. We recommend filling the tank up to the electrodes prior to inserting the gel cassettes. Complete filling of the tank not only prevent leaks from the inside to the outside but also acts as a heat sink.
- Not removing the tape from one of the gel cassettes. Make sure that the tape is removed from both gel cassettes before inserting them into the tank.
- Not placing the gel cassette all the way to the bottom of the tank prior to clamping it (this will create a bypass of the current at the bottom, so the gel will run slower or not run at all).

Answer Id: E11003

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Product FAQ

How can I store my viral stock?

Answer

If the medium is serum-free, add serum to 10%. Serum proteins act as substrates for proteases and therefore prevent degradation of viral coat proteins. Store viral stocks at 4 degrees C, and protect from light. Aliquots can be stored at -80 degrees C, but viral titer should be checked before use, as freeze/thaw cycles of the virus can result in a 10- to 100-fold decrease in viral titer.

Answer Id: E9402

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Product FAQ

What are the characteristics of TEMED (Cat. No. 17919)?

Answer

>Chemical name: N,N,N',N'- tetramethylethane-1,2-diamine
-Formula: C6H16N2
-CAS number: 110-18-9
-Molecular weight: 116.21-Purity: ≥ 99%
-Refractive index: 1.417 to 1.419
-Boiling range: 119 to 121 degrees C
-Clear liquid at room temperature, sold as a 25 mL volume in an amber bottle

Answer Id: E13367

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Product FAQ

Can I reuse the ProBond™ resin?

Answer

ProBond™ resin can be used for up to three or four purifications of the same protein without recharging. We only recommend reusing the resin for purification of the same recombinant protein. If you want to reuse the resin, wash it with 0.5 M NaOH for 30 minutes and equilibrate the resin with the appropriate binding buffer.

Answer Id: E12942

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Product FAQ

My Midi gel run in the XCell4 SureLock™ Midi-Cell is taking longer than usual. Can you please offer some tips?

Answer

Here are possible causes and solutions:

- Voltage is set too low. Set correct voltage as described in the manual.
- Buffers are too dilute. Check buffer recipe; re-make if necessary.
- Upper buffer chamber is leaking. Make sure the buffer core is firmly seated. If you are using the buffer dam, make sure the dam is properly positioned in the core. If the core gasket is damaged, replace with a fresh gasket. Check to ensure that the XCell4 SureLock™ Assembly is in the locked position.

Answer Id: E11004

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Product FAQ

Which lipid transfection reagent do you recommend for my cell line?

Answer

The best transfecting agent and efficiency would depend on the particular cell line you have. Please visit our online selection tools to see our recommendation for your specific cell line. If your cell line is not on the list, we recommend you try Lipofectamine™ LTX or Lipofectamine™ 2000 for plasmid transfection, and Lipofectamine™ RNAiMAX for siRNA transfection. For primary cells, Lipofectamine™ LTX with PLUS™ reagent is generally the best choice for plasmid transfection. For some hard-to-transfect cells, like suspension cells and stem cells, the Neon™ electroporation system usually works better compared to lipid transfection reagent.

Answer Id: E4502

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Product FAQ

Can I scale-up the production of recombinant protein using the baculovirus expression system? If so, what methods are available?

Answer

Yes, large-scale expression experiments can be performed. Please see below for different large-scale methods, requirements, added benefits, and references:
- Stirred bioreactor
- Airlift fermentor
- Insect larvae

Answer Id: E9403

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Product FAQ

Is there a visible sign that my cells are activated after incubating with CD3/CD28-coated Dynabeads™ magnetic beads?

Answer

Yes, when T cells are incubated with Dynabeads™ magnetic beads, they typically form visible cell-bead clusters in the wells within 12 hours. After 2-3 days the cells enter the blast stage (cell division) where they increase considerably in size and their morphology changes to a more stretched appearance. After expansion (days 8-12), they will regain their usual morphology of round and clearly defined cells.

Answer Id: E12152

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Product FAQ

I am getting a broad 50-68 kDa band across the entire length of the gel when I stain with SYPRO™ Ruby Protein Gel Stain and other protein stains. What is this and how can I prevent it?

Answer

Your samples or the gel wells were contaminated with keratins from skin or hair. Rinse out the gel wells with ultrapure water or running buffer before loading samples. Wear a lab coat and gloves when preparing samples and use microfuge tubes that have been stored in sealed plastic bags, not left out on the bench top, for preparing samples.

Answer Id: E11279

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Product FAQ

What are the Quality Specifications for each lot of TEMED?

Answer

The Certificate of Analysis lists actual test results for each lot and the minimum specification requirements:
-Purity > 99.0%
-Refractive index (nD20) between 1.417-1.419
-Boiling Range: 119-121 degrees C
-The solution must be a clear liquid, free of particulate matter

Answer Id: E13368

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Product FAQ

Can I recharge the ProBond™ resin?

Answer

Please keep in mind, while the ProBond™ resin is rechargeable, the Sepharose™ medium will sustain wear. If the resin turns white due to the loss of nickel ions from the column, recharge the resin.

To recharge 2 mL of resin in a purification column:

1.Wash the column two times with 8 mL 50 mM EDTA to strip away the chelated nickel ions.
2. Wash the column two times with 8 mL 0.5 M NaOH.
3. Wash the column two times with 8 mL of sterile, distilled water.
4. Recharge the column with two washes of 8 mL NiCl2 hexahydrate at a concentration of 5 mg/mL prepared in sterile, distilled water.
5. Wash the column two times with 8 mL distilled water.
6. Add 0.02% azide or 20% ethanol as a preservative and cap or apply a Parafilm™ cover to the column. Store at room temperature.

Answer Id: E12943

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Product FAQ

I am getting a “Short Circuit” error on my PowerEase™ 90W Power Supply? What should I do?

Answer

Here are possible causes and solutions:

- Load exceeds 500 mA. The output maximum on the power supply is 500 mA, so make sure that it is not exceeded.
- Blown fuse in the power supply. Replace fuse following instructions on Page 17 of the manual.
- Incorrect input voltage. Check input voltage switch near power inlet.

Answer Id: E11022

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Product FAQ

How does a two-temperature protocol work and when would you suggest using one?

Answer

You may choose to do a two-temperature protocol when the annealing temperature is relatively high. In this case, you would combine the annealing and the elongation steps, i.e., both can occur together at a temperature >62 degrees C. The advantage of a two-temperature protocol is that it is considerably quicker in comparison to the conventional three-temperature protocol.

Answer Id: E7274

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