After addition of the agarose overlay to my cells, they look like crescents or are granular. What happened?

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Answer

The agarose overlay was too hot. After addition of the agarose overlay, cells should still be round and healthy.

Answer Id: E9469

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I performed my PCR and see different banding patterns. What do the different PCR sizes mean?

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Answer

The expected PCR size will depend on the pFastBac™ vector that got transposed into the bacmid:

- Bacmid alone: 300 bp
- With pFastBac™ 1: 2300 bp + size of insert
- With pFastBac™ 1-Gus: 4200 bp
- With pFastBac™ HT: 2430 bp + size of insert
- With pFastBac™ HT-CAT: 3075 bp
- With pFastBac™ Dual: 2560 bp + size of insert
- With pFastBac™ Dual-Gus/CAT: 5340 bp
- With pDEST8: 2316 bp + size of insert
- With pDEST10: 2484 bp + size of insert
- With pDEST20: 3031 bp + size of insert

Answer Id: E9414

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What might cause plates to turn blue along with blue plaques?

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Answer

There are a few things that can turn plates blue:

- Too much virus when plating. Try a higher dilution.
- Cells are being singed when plated with hot melted agarose. This lyses the cells and releases lacZ into the agarose, turning it blue. Double-check plating temperatures. If plates are too wet, the blue can diffuse.

Answer Id: E9470

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How stable are your cationic lipids that are used in transfection?

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Answer

All the following lipids are stable at 4º C for at least one year:
11668-019 Lipofectamine™ 2000
10362-100 Cellfectin™ II
10459-014 DMRIE-C
10964-013 Lipofectamine™ PLUS™
18292-011 Lipofectin™
18324-012 Lipofectamine™

Answer Id: E3211

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Do you offer Gateway™ vectors for expression in plants?

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Answer

We do not offer any Gateway™ vectors for expression in plants.

Answer Id: E9854

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I forgot to add Bluo-gal to my plates. Can I add it later?

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Answer

On the day you intend to pick plaques, make a solution of Bluo-gal in DMSO at 20 mg/mL. Add 50 μL per plate and spread with a glass spreader under sterile conditions. Wait 30-60 min, and your plaques should turn blue.

Answer Id: E9471

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I’m getting mostly white/wild-type plaques instead of blue/recombinant plaques. What am I doing wrong?

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Answer

This is typically an indication of poor homologous recombination. Check the plasmid/linear DNA ratio you used. If there are some blue plaques, however, expand those viruses and check for their protein. In our experience, they are correct, even if they were in relatively low abundance.

Answer Id: E9474

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Can PCR primers be tailed directly with attL sites for direct recombination into the destination vector?

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Answer

No, this is not really feasible due to the fact that the attL sequence is approximately 100 bp, which is too long for efficient oligo synthesis. Our own maximum sequence length for ordering custom primers is 100 nucleotides. In contrast, the attB sequences are only 25 bp long, which is a very reasonable length for adding onto the 5' end of gene-specific PCR primers.

Answer Id: E3212

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Will increasing the Gateway™ cloning reaction time improve recombination efficiency?

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Answer

Yes, increasing the incubation time from 1 hour to 4 hours will generally increase colony numbers 2-3 fold. An overnight incubation at room temperature will typically increase colony yield by 5-10 fold.

Answer Id: E3206

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Can I purchase the 5X LR Clonase™ buffer or 5X BP Clonase™ buffer separately?

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Answer

We do not offer the 5X LR Clonase™ buffer and 5X BP Clonase™ buffer as standalone products. They are available as part of the enzyme kits.

Answer Id: E9855

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What is the purpose of the Proteinase K step following a Gateway™ LR Recombination reaction, and is it critical to the results?

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Answer

When the LR reaction is complete, the reaction is stopped with Proteinase K and transformed into E. coli resulting in an expression clone containing a gene of interest. A typical LR reaction followed by Proteinase K treatment yields about 35,000 to 150,000 colonies per 20ul reaction. Without the Proteinase K treatment, up to a 10 fold reduction in the number of colonies can be observed. Despite this reduction, there are often still enough colonies containing the gene of interest to proceed with your experiment, so the Proteinase K step can be left out after the LR reaction is complete if necessary.

Answer Id: E4230

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What does viral infection look like in early, late, and very late stages?

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Answer

Please see the description below of the different stages of viral infection:

Early
- Increased cell diameter-a 25-50% increase in the diameter of the cells may be observed.
- Increased size of cell nuclei-the nuclei may appear to "fill" the cells.

Late
- Cessation of cell growth-cells appear to stop growing when compared to a cell-only control.
- Granular appearance
- Signs of viral budding-vesicular appearance of cells.
- Viral occlusions-few cells will contain occlusion bodies, which appear as refractive crystals in the nucleus of the insect cell.
- Detachment-cells release from the dish or flask.

Very late
- Cell lysis-a few cells may fill with occluded virus, die, and burst, leaving signs of clearing in the monolayer.

Answer Id: E9400

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How large can PCR fragments be and still be cloned into a Gateway™ Entry vector?

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Answer

There is no size restriction on the PCR fragments if they are cloned into a pDONR vector. The upper limit for efficient cloning into a TOPO™ adapted Gateway™ Entry vector is approximately 5 kb. A Gateway™ recombination reaction can occur between DNA fragments that are as large as 150 kb.

Answer Id: E3224

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I’m worried that I am not getting plaques. How many days does it take to see plaques and what size are they typically?

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Answer

Normally, very small white dots show up about 5-7 days and 1 mm plaques show up around day 10. Plaques can vary in size from 1 mm to 4 mm.

Answer Id: E9472

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How clean must my DNA be to use in a Gateway™ cloning reaction?

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Answer

Mini-prep (alkaline lysis) DNA preparations work well in Gateway™ cloning reactions. It is important that the procedure remove contaminating RNA for accurate quantification. Plasmid DNA purified with our S.N.A.P.™ nucleic acid purification kits, ChargeSwitch™ kits, or PureLink™ kits are recommended.

Answer Id: E3204

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