Product FAQ

Can I use the pENTR™/H1/TO or Lenti4/BLOCK-iT™-DEST for constitutive expression?

Answer

Yes, as long as you do not use a cell line expressing the Tet repressor, expressing will be constitutive.

Answer Id: E10024

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Product FAQ

I’m not getting gene knockdown after tetracycline induction for my pENTR™/H1/TO construct or Lenti4/BLOCK-iT™-DEST construct experiment. What do you suggest I try?

Answer

The shRNA chosen may not be working. Verify that the shRNA sequence does not contain more than 3 tandem Ts which can cause premature transcription termination. You can try to select a different target region. Check the hairpin design for the shRNA. Ensure that the correct amounts of tetracycline were added. Cells should be treated 3-24 hours after transfection with tetracycline to induce shRNA expression. Assay for the target gene knockdown 24-96 hours following induction. Lastly, please check that the construct was transduced into a T-REx™ repressor-expressing cell line. (To create your own cell line that stably expresses the Tet repressor, use either pcDNA™6/TR (for your pENTR™/H1/TO construct) or pLenti6/TR (for your Lenti4/BLOCK-iT™-DEST construct) and maintain the cell line in medium containing blasticidin.)

Answer Id: E10025

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Product FAQ

I’m getting differently sized colonies after TOP10 E. coli transformation when using the miRNA lentiviral expression system. Which one should I use?

Answer

Some transformants may contain plasmids in which unwanted recombination has occurred between the 5’ and 3’ LTR. We recommend using our One Shot™ Stbl3™ Chemically Competent E. coli cells, as they help in stabilizing lentiviral DNA containing direct repeats, and generally give rise to fewer unwanted recombinants. We recommend screening both colony sizes; however, in general, for lentiviral plasmids the small colonies tend to be the correct clones. Large colonies may have undergone a recombination event to delete part of the plasmid, thus allowing the cells to grow faster.

Answer Id: E10026

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Product FAQ

I labeled my protein with your kit, but now it‘s too dilute for my application. What should I do?

Answer

There are different ways to concentrate your protein. These include:

Centrifugation (filtration ) using concentrators
Dialysis
Lyophilization
Precipitation

Answer Id: E12879

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Product FAQ

I’m getting few or no colonies, even with the transformation control. What could be the cause of this?

Answer

Ensure that the competent cells used were stored properly at -80 degrees C, and thawed on ice for immediate use. When adding DNA, mix competent cells gently: do not mix by pipetting up and down. Also do not exceed the maximum recommended amount of DNA for transformation (100 ng) or allow the volume of DNA added to exceed 10% of the volume of the competent cells, as these may inhibit the transformation.

Answer Id: E10027

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Product FAQ

What do you recommend for resealing resuspended siRNA stock plates?

Answer

For those who want to re-seal plates after resuspension, we recommend aluminum seals over plastic. We have used the following seal in the past with good success:

- Thermo Scientific™ Nunc™ Microplate Lids; Thermo Scientific Cat. No. 250002
- Sealing Films, Axygen Scientific, (Cat. No. 47734-816), Description: Aluminum Sealing Film. Maintains excellent seal on microplates under the widest temperature conditions ( -80° to 97 degrees C) and reagents. Ideal for light-sensitive samples. Uniformly applied adhesive.

Answer Id: E10044

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Product FAQ

I see that you offer multiple immobilization resins for protein immobilization. Which one should I use?

Answer

Please view our selection chart (http://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-purification-isolation/protein-purification/protein-immobilization.html) to choose the right immobilization solution for your experiment.

Answer Id: E12880

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Product FAQ

I am not getting any colonies after titering. What would suggest I try?

Answer

Perform a kill curve to determine the antibiotic sensitivity of your cell line. Ensure that viral stocks are stored properly at -80 degrees C, and do not undergo freeze/thaw more than 3 times. Lastly, transducer the lentiviral contruct into cells in the presence of Polybrene™ reagent.

Answer Id: E10028

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Product FAQ

How much Lipofectamine™ RNAiMAX reagent is required for a genome-wide screen?

Answer

It is expected that 800 plates of 96 wells would be used for a genome-wide screen. Allowing for 3 biological replicates, the total number of wells to be transfected are: 800 plates x 96 wells x 3 replicates = 230,400 wells.
0.15 μL of Lipofectamine™ RNAiMAX reagent needs to be added per well = 34,560 μL of Lipofectamine™ RNAiMAX reagent= approximately 35 mL total of Lipofectamine™ RNAiMAX reagent for a single genome-wide screen with 3 replicates.

Answer Id: E10045

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Product FAQ

How do your AminoLink™ Coupling Resin and AminoLink™ Plus Coupling Resin work?

Answer

AminoLink™ and AminoLink™ Plus Supports are activated with an aldehyde group to form a Schiff base, which is reduced to a stable, non-reversible secondary amine. Coupling efficiency often exceeds 85% with this support.

Answer Id: E12881

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Product FAQ

I’m seeing nonspecific, off-target gene knockdown. What should I do?

Answer

The target sequence used may contain strong homology to other genes; please select a different target region.

Answer Id: E10029

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Product FAQ

At what siRNA concentration should I do my screen? Any other tips on transfection?

Answer

The efficiency with which mammalian cells are transfected with siRNA will vary according to cell type and the transfection agent used. This means that the optimal concentration used for transfections should be determined empirically. Since Silencer™ Select siRNAs exhibit superior silencing potency compared to other siRNAs, we suggest starting concentrations of 5- to 20-fold less than typically used for transfection of your experimental cell lines. We have found that Silencer™ Select siRNAs reduced mRNA levels >80% at final concentrations of 2-10 nM using lipid-mediated transfection in HeLa and U2OS human osteosarcoma cells.

Answer Id: E10046

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Product FAQ

What is the difference between Invivofectamine™ 2.0 Reagent and Invivofectamine™ 3.0 Reagent?

Answer

Invivofectamine™ 3.0 Reagent is an improved formulation with the following increased benefits:

- Lower toxicity
- Higher transfection efficiency with less siRNA; sustained knockdown up to 2 weeks
- Increased stability
- Shorter overall workflow; no dialysis or diafiltration needed

Answer Id: E10382

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Product FAQ

What is the difference between GlycoLink™ and CarboxyLink™ Gels?

Answer

GlycoLink™ gel is hydrazide-activated crosslinked beaded agarose, and it is useful for coupling glycoproteins via aldehydes formed from their sugars by sodium meta-periodate oxidation. Reaction of aldehydes with hydrazide- activated resin is catalyzed by aniline resulting in >90% coupling in 4 hours or less. CarboxyLink™ gel on the other hand is crosslinked beaded agarose activated with diaminodipropylamine (DADPA) and is useful for immobilizing carboxyl- containing biomolecules after EDC activation. Actually, both resins can be used with EDC to couple ligands via carboxylic acids. Note: Both immobilization chemistries are available on UltraLink™ Support as UltraLink™ Hydrazide and UltraLink™ DADPA respectively.

Answer Id: E12899

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Product FAQ

What is the difference between AminoLink™ and AminoLink™ Plus supports?

Answer

AminoLink™ Plus Coupling Resin is activated at a higher level and has higher flow rates than the original AminoLink™ Coupling Resin, resulting in higher capacity and faster purification. AminoLink™ Supports can be used to immobilize any molecule with a primary amine.

Answer Id: E12882

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