Promega eNotes - LabFacts

LABFACT #36

Consider the downstream application for your PCR product when selecting a purification method. If the analytical gel shows a single band of expected base pair size, the amplimer can be directly purified. If there are excessive primer dimers or additional bands visible, gel purification can be used to isolate the product of interest.

LABFACT #35

When expressing your protein in a cell-free system, make sure the combination of promoter and DNA conformation is appropriate. For example, the T7 RNA polymerase promoter may express better in a linearized template while the SP6 promoter transcribes better in a circular DNA.

LABFACT #34

Be sure to dephosphorylate your linear vector if it was cut by a single restriction endonuclease or if the two restriction sites are close together on the plasmid. Dephosphorylation reduces the background of religated vector during cloning.

LABFACT #33

T7 RNA polymerase transcribes optimally at 37°C. In some cases, transcription at temperatures lower than 37°C may result in a higher proportion of full-length transcripts (1).
1. Krieg, P.A. (1990) Nucleic Acids Res. 18, 6463.

LABFACT #32

Vary the incubation time and temperature for successful ligation. Blunt-end ligations benefit from lower temperatures (4–16°C) and longer incubation times (4 hours to overnight). Cohesive-end ligations can generally be performed at higher temperatures (15–20°C) with shorter incubation times (3–16 hours).

LABFACT #31

We recommend using a 1:1, 1:3 or 3:1 molar ratio of vector:insert DNA when cloning a fragment into a plasmid vector. These ratios will vary with other types of vectors (for example, cDNA and genomic cloning vectors).

LABFACT #30

GST-tagged proteins can be purified from mammalian cells with reduced specificity. Since mammalian cells contain GST (1), the endogeneous GST will compete for binding on the purification substrate such as the MagneGST™ Glutathione Particles.
1. Hayes, J.D. and Pulford, D.J. (1995) Crit. Rev. Biochem. Mol. Biol. 30, 445–600.

LABFACT #29

Bioluminescent assays can be 10- to 1,000-fold more sensitive than fluorescent assays. More information on bioluminescence vs. fluorescence.

LABFACT #28

TSAP Thermosensitive Alkaline Phosphatase (Cat.# M9910) is effectively and irreversibly inactivated by heating at 74°C for 15 minutes. Therefore, a DNA cleanup step is not required before ligation.

LABFACT #27

To ensure efficient ligation, do not frequently freeze and thaw the Ligase 10X Buffer supplied with Promega T4 DNA Ligase. ATP is sensitive to freeze-thaw cycles. We recommend storing the buffer in small aliquots.

LABFACT #26

G418 blocks protein synthesis in mammalian cells by interfering with ribosomal function. It is an aminoglycoside, similar in function to neomycin, gentamycin and kanamycin.

LABFACT #25

When using T4 DNA Polymerase to fill in a 5´ overhang, do not allow the reaction to proceed more than 90 minutes. As the dNTPs in the reaction are depleted, T4 DNA Polymerase will begin degrading the double-stranded template.

LABFACT #24

When mixing protein-containing solutions, be sure to mix gently. Vortexing can result in protein denaturation due to exposure to oxygen at the liquid surface. Reference: Basic Biochemical Methods (1993) Alexander, R.R. and Griffiths, J.M. John Wiley & Sons, Inc. New York.

LABFACT #23

For long-term storage of genomic DNA, keep the DNA in a buffer like TE Buffer or DNA Rehydration Solution (Cat.# A7963). Long-term storage of gDNA in water can lead to autolytic degradation.

LABFACT #22

When fixing cells, methanol-free formaldehyde can be substituted for paraformaldehyde; both work equally well. The formaldehyde must be methanol-free, as methanol is often added to formaldehyde to prevent polymerization.

LABFACT #21

The tetrazolium salts, MTT and MTS, are commonly used to determine viability of mammalian cells. However, these dyes can also be used to monitor viability of yeast, plant and even bacterial cells.

LABFACT #20

Adding chloramphenicol or spectinomycin to a bacterial culture can help increase yields of low-copy-number plasmids but will not noticeably increase yields of high-copy-number plasmids.

LABFACT #19

During an in vitro transcription reaction to generate radiolabeled RNA probes, the addition of 0.05% Tween^® can help increase the activity of the RNA polymerase.

LABFACT #18

When performing RNase protection assays, the use of RNase ONE™ Ribonuclease enables more accurate mapping than RNase A and Rnase T1. RNase ONE™ Ribonuclease cleaves 3′ of any nucleotide, while RNase A cleaves 3′ of only C and A residues and RNase T1 cleaves 3′ of G residues.

LABFACT #17

Enzymes that are not heat-inactivated can be inactivated by diethyl pyrocarbonate (DEPC) treatment. Add DEPC to a final concentration of 0.1% and incubate the reaction at 65°C for 30 minutes with the cap open during the last 15 minutes to vent the gases.

LABFACT #16

More restriction enzyme is generally required to digest supercoiled DNA than linear DNA. If you linearize supercoiled DNA with one restriction enzyme, less of a second restriction enzyme is required for complete digestion.

LABFACT #15

Plasmid DNA can be sequenced using PCR directly from E. coli (#TM024) or yeast colonies (Promega Notes 44).

LABFACT #14

When isolating plasmid from bacterial cells for use in eukaryotic transfection experiments, be sure to use a protocol that limits the amount of endotoxin in the preparation. For more information, see Technical Bulletins #TB259 and #TB314.

LABFACT #13

When making RNase-free solutions, do not treat Tris buffers with DEPC. The DEPC reacts rapidly with amines. For more information on creating an RNase-free environment see the RNA Analysis Notebook #BR120.

LABFACT #12

Large full-length transcripts (27.3kb) can be produced in minutes using the T7 RiboMAX™ Express Large-Scale RNA Production System (more...).

LABFACT #11

New restriction enzyme isoschizomers often are less expensive or offer performance advantages over the classic enzymes. For more information on the use of isoschizomers and neoschizomers, see the Promega Notes article, "Making the Cut...".

LABFACT #10

The 3´ to 5´ exonuclease activity of T4 DNA Polymerase (Cat.# M4211, M4215) is often used to remove single-stranded 3´ overhangs from DNA fragments. The presence of 100μM dNTPs in the reaction will prevent T4 DNA Polymerase from degrading double-stranded DNA and create a blunt end. To read more, see FAQ#025.

LABFACT #9

Promega's Technical Manuals and Bulletins, Product Information Sheets, and Protocol Cards are an excellent source of technical information and are available online at www.promega.com/tbs/.

LABFACT #8

PCR products created using Taq, Tth or Tfl DNA Polymerase often have a 3´-A overhang and can be cloned using the pGEM^®-T and pGEM^®-T-Easy Vector Systems.
(from #TM042)

LABFACT #7

The Apo-ONE™ Homogeneous Caspase-3/7 Assay may be used with purified enzyme preparations, cell extracts, or cultures of adherent, suspension or primary cells.
(from #TB295)

LabFact #6

For the Dual-Luciferase^® Reporter Assay System, only use Passive Lysis Buffer since PLB is specially formulated to minimize background autoluminescence.
(from #TM040)

LabFact #5

The pGEM^®-T Easy Vector System protocol uses the 2X Rapid Ligation Buffer. Use the appropriate volume when setting up the ligation reaction.
(from #TM042)

LabFact #4

ImProm-II™ Reverse Transcription reaction conditions support PCR amplification. No dilution of the cDNA is necessary. Add heat-inactivated reverse transcription products directly to the PCR mix.
(from #TM236)

LabFact #3

DNA templates for ABI PRISM^® BigDye™ Terminator chemistry include plasmid DNA, PCR and RT-PCR products, m13DNA or Lambda DNA.

LabFact #2

RT-PCR should be performed in the absence as well as presence of the reverse transcriptase, to assess DNA contamination in the template RNA. In addition, "no-template" negative control reactions should always be performed.

LabFact #1

We highly recommend the use of barrier pipet tips for assembling components for PCR or RT-PCR to prevent contamination of reactions and reagents with template DNA, RNA or primers.

LabFacts

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