Product FAQ

Which library files are needed for the miRNA 1.0, 2.0 and 3.0 Arrays?

Answer

Array Type: Library File
GeneChip™ miRNA Array: miRNA 1_0_2Xgain
GeneChip™ miRNA 2.0 Array: miRNA 2_0
GeneChip™ miRNA 3.0 Array: miRNA 3_0

Answer Id: E14197

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Product FAQ

When blotting with the XCell II™ Blot Module, what would happen if I filled the outer buffer chamber with transfer buffer instead of water?

Answer

This is perfectly acceptable with the XCell II™ Blot Module. The liquid in the outer buffer chamber only serves as a coolant or heat sink. The reason why water is recommended is because it is a less expensive alternative.

Answer Id: E3280

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Product FAQ

What software package is necessary for array QC analysis?

Answer

Array Type: Software
GeneChip™ miRNA 1.0 Array: miRNA QC Tool Version 1.0.33.0
GeneChip™ miRNA 2.0 Array: miRNA QC Tool Version 1.1.1.0/Expression Console 1.2 or higher
GeneChip™ miRNA 3.0 Array: Expression Console 1.2 or higher

Answer Id: E14198

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Product FAQ

I am getting low absorbance readings in all the wells of my ELISA plate although the wells appear well developed. What causes this and how can it be fixed?

Answer

Incorrect wavelength used for reading can give a low absorbance reading. Adjust the wavelength setting on the plate reader to the optimal wavelength for the substrate being employed.

Answer Id: E3972

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Product FAQ

Do you have any tips to improve results when Western transferring two gels at the same time?

Answer

Less-than-optimal transfer in the second gel is not uncommon and frequently requires adjustments in protocol. To achieve similar transfer efficiencies in the two gels you can transfer them one at a time or consider the following suggestions:

Make sure that the blot module is not overfilled with buffer, i.e., fill to point where pads are just covered and no higher. If the buffer level is too high, some current will bypass the gels. If it is overfilled, shorting, arcing, and other problems can occur.

Use antioxidant when transferring reduced proteins.

Increase transfer time by 30 to 60 minutes. The longer run time allows the slower moving proteins time to move out of the gel.

As proteins are being driven out of the gel by the SDS on their surface, it is important that enough is retained to keep them mobile. However, the methanol that is included in the transfer buffer will remove some of the SDS, and the higher the methanol concentration, the more SDS is washed off of the protein. Methanol is included in order to increase binding to nitrocellulose membranes via hydrophobic interactions. However, if using PVDF or nylon membranes, less or even no methanol may be required. 20% methanol is a good starting point for most situations In the event that the transfer is less than ideal, methanol levels should be reduced by at least 50%. The optimal percentage should be determined empirically. Something to keep in mind is that with increased mobility, the proteins may just move straight though the first membrane. In this case, a second membrane may trap these samples.

It is possible to BRIEFLY (1 min) soak the second gel in transfer buffer with 0.01 to 0.02% SDS before assembling the sandwich. This must be a short soak or the proteins will tend to diffuse out of the gel. Adding 0.01-0.02% SDS to the transfer buffer and using it for the transfer will also aid in protein mobility.

Halfway through the transfer process (30 min), swap the front gel and back gel in the blotter. This is fairly easy to do if you just swap pad-paper-gel-membrane-pad sections.

Another recommendation: if you must transfer simultaneously, and want to be able to compare results of both blots, use an internal control in both gels to get an idea of the relative transfer efficiency and to afford some normalization of the results.

Answer Id: E3281

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Product FAQ

What media do you recommend for culturing Human Corneal Epithelial Cells (HCEC)?

Answer

We recommend culturing HCECs in keratinocyte serum-free medium (KSFM), as the cells are manufactured and quality-controlled using this medium. However, if a defined growth system is preferred, we recommend defined keratinocyte serum-free medium (Defined Keratinocyte-SFM). Note that a Coating Matrix Kit is required for culturing HCECs in Defined Keratinocyte-SFM. In-house testing has shown comparable HCEC growth and morphology in both KSFM and Defined Keratinocyte-SFM.

Answer Id: E11974

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Product FAQ

What workflow is recommended in Expression Console v1.2 or higher?

Answer

To evaluate the success of the labeling protocol and array processing, open line graphs for the spike-in labeling control probe sets in Expression Console. Once the RMA+DABG analysis is complete, click on the icon for line graphs or select Graph > Line Graph - Report Metrics and select the check boxes for desired probe sets. If the labeling protocol was successful, the following spike-in control probe sets (representing synthetic miRNAs present in vial 8) should have signal greater than or equal to 1000 (or 9.96 for log2 signal):

spike_in-control-2_st
spike_in-control-23_st
spike_in-control-29_st
spike_in-control-31_st
spike_in-control-36_st
Oligos 2, 23 and 29 are RNA, and confirm the poly(A) tailing and ligation. Oligo 31 is poly(A) RNA and confirms ligation. Oligo 36 is poly(dA) DNA and confirms ligation and the lack of RNases in the RNA sample.

Answer Id: E14199

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Product FAQ

Is a sample size available for MagJET kits?

Answer

No, we do not offer a sample size for MagJET kits.

Answer Id: E8855

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Product FAQ

What are M13-tailed primers used for in DNA sequencing and can I have the sequence for these primers?

Answer

The M13-tailed primers are used to simplify the workflow when sequencing PCR products and they reduce the loss of the 5' unresolvable bases. When the PCR primers contain M13 tails on their 5' ends, the M13 sequence is incorporated into the amplicons. This enables the use of sequencing master mixes containing either the universal M13 forward or M13 reverse primers. The sequence for the M13 forward and reverse primers are as follows:

-M13 forward primer sequence: 5′ TGTAAAACGACGGCCAGT 3′
-M13 reverse primer sequence: 5′ CAGGAAACAGCTATGACC 3′

Answer Id: E15319

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Product FAQ

Sometimes my positive population appears as a tight doublet when using the eBioscience™ OneComp eBeads. What should I define or gate as positive?

Answer

There is some antibody-based variation on the shape of the positive peak. When the positive peak appears as a doublet, it is appropriate to gate on the entire positive population. Appropriate compensation values will typically result from this strategy.

Answer Id: E14462

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Product FAQ

I am not getting reproducible results when I blot multiple gels simultaneously. What causes this and what suggestions do you have?

Answer

Transferring multiple gels simultaneously in the same apparatus rarely results in equivalent transfer efficiencies for all of the gels. You can either transfer gels one at a time, or, if you must transfer simultaneously, use an internal control in both gels to get an idea of the relative transfer efficiency and to allow you some normalization capability.

Answer Id: E3974

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Product FAQ

What is the 54 kDa band observed in human tissue lysates when I use WesternBreeze™ kits?

Answer

A number of human alkaline phophatases that could be consuming the WesternBreeze™ substrate have a molecular weight close to 50 kDa. These would be detected as a band of approximately 54 kDa. These proteins are known to dimerize, and can show as a band of approximately 110 kDa in native gels.

Answer Id: E3282

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Product FAQ

I used the iBind™ Flex Western system and my membrane is very “spotted”. What could have caused this?

Answer

Here are possible causes and solutions:

- Poor or incomplete transfer: Repeat blot.
- Membrane pads are dirty or contaminated: Soak with detergent and rinse thoroughly with purified water before use. Replace pads when they become worn or discolored.
- Membrane not completely wet: Follow instructions for prewetting the membrane.
- Membrane is contaminated by fingerprints or keratin protein: Wear clean gloves at all times and use forceps when handling membranes. Always handle membranes around the edges.
- Uneven blocking: The incubation dish must be small enough to allow thorough coverage of membrane.
- Ink used to label membrane: Any labeling of the membrane with ink should be limited to the low MW region of the blot.
- iBind™ Flex Card damaged: Replace with new card. Ensure that rolling of the membrane on the card is limited to membrane region.
- Membrane is not in proper contact with the iBind™ Flex Card: Place the membrane on the iBind™ Flex Card immediately after adding a 1 mL pool of 1X iBind™ Flex Solution/ iBind™ Flex FD Solution. Use the roller provided to ensure proper contact.

Answer Id: E11317

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Product FAQ

Do Human Corneal Epithelial Cells (HCEC) require feeder cells or matrix coatings for proliferation?

Answer

Feeder cells are not needed for culturing HCECs using KSFM or Defined Keratinocyte-SFM. However, HCECs cultured in Defined Keratinocyte-SFM require the use of a Coating Matrix Kit (recombinant type I collagen) for efficient attachment and growth.

Answer Id: E11975

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Product FAQ

Which arrays are supported by Command Console Software?

Answer

All current, catalog arrays are supported by Command Console Software.

Answer Id: E14216

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