Can I sequence a peptide that is acetylated or biotinylated?

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No. These groups effectively block the N terminus.

Answer Id: 1267

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b51a15f382ac914391a58850ab343b00_FAQ

How does AmpliTaq Gold® DNA Polymerase differ from AmpliTaq® DNA Polymerase?

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AmpliTaq Gold® DNA Polymerase is a modified form of AmpliTaq® DNA Polymerase that contains a proprietary chemical (or so-called hot start molecule) bound to the enzyme's active site. In order to activate the AmpliTaq Gold® DNA Polymerase fully, we recommend an initial activation step of 95 degrees C for 10 min when using GeneAmp® 10X PCR Buffer I and/or GeneAmp® 10X PCR Buffer II and Mg in one of our thermal cyclers. When using GeneAmp® 10X PCR Gold Buffer, activation time can be reduced to 5 minutes. Once activation is complete, you can proceed with your standard PCR cycling program (denaturing, annealing, extension, etc).

Answer Id: 1339

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d10ec7c16cbe9de8fbb1c42787c3ec26_FAQ

What is Gateway® Cloning Technology?

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Gateway® Cloning Technology is an easy-to-use system for cloning and subcloning DNA segments (e.g. genes of interest), facilitating gene functional analysis, protein expression, and the integration of technology platforms. One can also readily clone PCR products into so-called Gateway® "Entry" vectors. To shuttle inserts from one vector to another, the Gateway® Cloning Technology uses bacteriophage lambda-based site-specific recombination. There is no need to use restriction enzymes and ligase to subclone inserts.

One advantage of Gateway® Cloning Technology is that genes present in a single Gateway® Entry vector can be subcloned into multiple different Gateway® Destination vectors. After this 1 hour in vitro subcloning reaction, a high percentage of the colonies obtained carry the desired expression clone. For more details, please see the product manual for cat# 12535029 or cat# 12535037.

Answer Id: 3029

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a0ba2648acd23dc7a5829968ce531a7d_FAQ

Which strand is produced using M13K07 to make ssDNA from pPROEX HT or pFastBAC™ vectors?

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You will get the antisense strand with pProEXHT.
You will get the + or sense strand with pFastBac I of pFastBac™ HT.

Answer Id: 3167

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20c86a628232a67e7bd46f76fba7ce12_FAQ

What primer purity should be used for adding attB sites to my PCR product?

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Standard desalted purity is generally sufficient for creating attB primers. We examined HPLC-purified oligos for Gateway® cloning (about 50bp long) and found only about a 2-fold increase in colony number over standard desalted primers. If too few colonies are obtained, you may try to increase the amount of PCR product used and/or incubate the BP reaction overnight.

Answer Id: 3172

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e25cfa90f04351958216f97e3efdabe9_FAQ

What is the smallest fragment that can be used in a Gateway® reaction?

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The smallest size we have recombined is a 70 bp piece of DNA located between the att sites. Very small pieces are difficult to clone since they negatively influence the topology of the recombination reaction.

Answer Id: 3198

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b08354f3688c4e4e8c52c207d7d5b8c3_FAQ

Can the attB primers anneal in a non-specific manner?

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No, attB primers are highly specific under standard PCR conditions. We have amplified from RNA (RT-PCR), cDNA libraries, genomic DNA, and plasmid templates without any specificity problems.

Answer Id: 3199

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b59307fdacf7b2db12ec4bd5ca1caba8_FAQ

Will Gateway® att sites affect the expression of my protein?

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Expression experiments have shown that the extra amino acids contributed by the attB site to a fusion protein will most likely have no effect on protein expression levels or stability. In addition, they do not appear to have any effect on two-hybrid interactions in yeast. However, as is true with the addition of any extra sequences that result from tags, the possible effects will be protein-dependent.

Answer Id: 3200

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731ae30af8750c2d28720ea3c1f8c2b1_FAQ

Are the Gateway® attB1 and attB2 sites the same as the attB site used for recombination into E. coli by bacteriophage lambda?

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The Gateway® attB sites are derived from the bacteriophage lambda site-specific recombination, but are modified to remove stop codons and reduce secondary structure. The core regions have also been modified for specificity (i.e., attB1 will recombine with attP1 but not with attP2).

Answer Id: 3201

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24ec8468b67314c2013d215b77034476_FAQ

Where is the ATG relative to the 5' attB site in a Gateway® expression clone?

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This depends on whether you are expressing a fusion or a native protein in the Gateway® destination vector. For an N-terminal fusion protein the ATG will be given by the destination vector and it will be upstream of the attB1 site. For a C-terminal fusion protein or a native protein, the ATG should be provided by your gene of interest, and it will be downstream of the attB1 site.

Answer Id: 3202

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7486cef2522ee03547cfb970a404a874_FAQ

How would you incorporate a leader sequence for secretion into an entry vector?

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A simple way to express a protein with a leader sequence is to have the leader sequence encoded in the destination vector. The other option is to have the leader sequence subcloned into the entry vector using restriction enzymes, or incorporate the leader sequence into the forward PCR primer when cloning a PCR product into the entry vector. Please see Esposito et al. (2005), Prot. Exp. & Purif. 40, 424-428 for an example of how a partial leader sequence for secretion was incorporated into an entry vector.

Answer Id: 3203

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799de6d3dae4c924142cf245a1d7f703_FAQ

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

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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: 3204

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640258597cbc50037072712f964cf5d8_FAQ

How many times can I thaw BP Clonase® II and LR Clonase® II?

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BP Clonase® II and LR Clonase® II can be freeze/thawed at least 10 times without significant loss of activity. However, you may still want to aliquot the enzymes to keep freeze/thaw variability to a minimum.

These enzymes are more stable than the original BP and LR Clonase® and can be stored at -20°C for 6 months.

Answer Id: 3205

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9ef2ed4b7fd2c810847ffa5fa85bce38_FAQ

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

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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: 3206

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211ed78fe91938b90f84a51944b08d5a_FAQ

What are the prerequisites for Gateway® cloning and expression?

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The gene of interest must be flanked by the appropriate att sites, either attL (100 bp) in an Entry clone or attB (25 bp) in a PCR product. For Entry clones, everything between the attL sites will be shuttled into the Gateway® destination vector containing attR sites, and a PCR product flanked by attB sites must be shuttled into an attP-containing donor vector such as pDONR™221.

The location of translation initiation sites, stop codons, or fusion tags for expression must be considered in your initial cloning design. For example, if your destination vector contains an N-terminal tag but does not have a C-terminal tag, the vector should already contain the appropriate translation start site but the stop codon should be included in your insert.

Answer Id: 3207

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