Zero Blunt™ PCR Cloning Kit
Zero Blunt™ PCR Cloning Kit
Invitrogen™

Zero Blunt™ PCR Cloning Kit

The Zero Blunt™ PCR Cloning Kit offers an easy method for high-efficiency (>80%) cloning of blunt-end PCR products amplified withRead more
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Catalog NumberQuantity
K27002020 Reactions
K27004040 Reactions
Catalog number K270020
Price (USD)
768.65
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808.00
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Quantity:
20 Reactions
Recurring order eligible. Learn more »
Price (USD)
768.65
Online Exclusive
808.00
Save 39.35 (5%)
Each
Add to cart
Ask our AI about this Product
The Zero Blunt™ PCR Cloning Kit offers an easy method for high-efficiency (>80%) cloning of blunt-end PCR products amplified with proof-reading, thermostable DNA polymerases. The Zero Blunt™ PCR Cloning Kit uses the multipurpose cloning vector pCR™-Blunt and ExpressLink™ T4 DNA Ligase to generate a ligation product in a five-minute, room-temperature ligation step.

Features of the Zero Blunt™ Cloning™ Kit with pCR™-Blunt vector:
Fast & convenient—5-minute, room-temperature ligation
EfficientccdB gene for positive selection results in >80% clones with correct insert
Flexible—choice of kanamycin or Zeocin™ resistance for flexible antibiotic selection

The pCR™-Blunt vector provides:
EcoR I sites flanking the PCR product insertion site for excision of inserts
• T7 promoter/primer site for in vitro RNA transcription and sequencing
• M13 forward and reverse primer sites for sequencing or PCR screening

How Zero Blunt™ PCR Cloning Works
The Zero Blunt™ PCR Cloning Kit is designed to clone blunt PCR fragments (or any blunt DNA fragment) with a low background of non-recombinants. The pCR™-Blunt vector contains the lethal E. coliccdB gene fused to the C-terminus of LacZα (Bernard et al., 1994). Ligation of a blunt PCR fragment disrupts expression of the lacZα-ccdB gene fusion permitting growth of only positive recombinants upon transformation. Cells that contain non-recombinant vector are killed when the transformation mixture is plated.

Kit Configurations
The Zero Blunt™ PCR Cloning Kit is offered in a variety of configurations: with One Shot™ TOPO10 Chemically Competent E. coli (K2700-20 and K2700-40) and without competent cells (K2750-20 and K2750-40) in 20- and 40- reaction kit sizes.
For Research Use Only. Not for use in diagnostic procedures.
Specifications
Bacterial or Yeast StrainTOP10
Cloning MethodBlunt PCR
Product LineZero Blunt™
Product TypePCR Cloning Kit
PromoterT7
Quantity20 Reactions
VectorBlunt DNA Cloning Vectors
FormatKit
Unit SizeEach
Contents & Storage
Zero Blunt™ PCR cloning kits contain linearized pCR™-Blunt vector, ExpressLink™ T4 DNA ligase , 5X ExpressLink™ T4 DNA ligase buffer, control template, dNTPs, sterile water, and M13 forward and reverse primers. Competent cell kits contain One Shot™ chemically competent E. coli, S.O.C. medium, and a supercoiled control plasmid.

Store the One Shot™ E. coli at -80°C. Store all other components at -20°C. All reagents are guaranteed stable for 6 months when properly stored.
Ready-to-use Bacterial Growth Media

Ready-to-use Bacterial Growth Media

See how these mediums can help with critical aspects of cloning!

Gibco LB Broth

Invitrogen S.O.C. Medium

Invitrogen One Shot LB Agar*

*Only available in North America and selected European countries

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Frequently asked questions (FAQs)

What is the best molar ratio of PCR product:vector to use for TOPO TA cloning? Is there an equation to calculate the quantity to use?

We suggest starting with a molar ratio of 1:1 (insert:vector), with a range of 0.5:1 to 2:1. The quantity used in a TOPO cloning reaction is typically 5-10 ng of a 2 kb PCR product.

Equation:

length of insert (bp)/length of vector (bp) x ng of vector = ng of insert needed for 1:1 (insert:vector ratio)

What is the best ratio of insert:vector to use for cloning? Is there an equation to calculate this?

The optimal ratio is 1:1 insert to vector. Optimization can be done using a ratio of 0.5-2 molecules of insert for every molecule of the vector.

Equation:

length of insert (bp)/length of vector (bp) x ng of vector = ng of insert needed for 1:1 insert:vector ratio

I'm seeing a lot of vector-only colonies when I try to perform a negative control reaction using vector only (no insert) for a TOPO reaction. Is my TOPO vector re-ligating?

Using the vector only for transformation is not a recommended negative control. The process of TOPO-adaptation is not a 100% process, therefore, there will be “vector only” present in your mix, and colonies will be obtained.

I'm trying to clone in my phosphorylated PCR product into a TOPO vector, and I'm getting no colonies. However, when I clone the same product into a TA vector, everything works perfectly. Why is this?

Phosphorylated products can be TA cloned but not TOPO cloned. This is because the necessary phosphate group is contained within the topoisomerase-DNA intermediate complex of the vector. TOPO vectors have a 3' phosphate to which topoisomerase is covalently bound and a 5' phosphate. Non-TOPO linear vectors (TA and Blunt) have a 3' OH and a 5' phosphate. Phosphorylated products should be phosphatased (CIP) before TOPO cloning.

I'm able to get a lot of colonies, however, none contain my insert of interest. What should I do?

You may be cloning in an artifact. TA and TOPO Cloning are very efficient for small fragments (< 100 bp) present in certain PCR reactions. Gel-purify your PCR product using either a silica-based DNA purification system or electroelution. Be sure that all solutions are free of nucleases (avoid communal ethidium bromide baths, for example.)

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Safety Data Sheets

Vector Information

Vector Name
Vector Map
Polylinker
Sequence
Restriction
pHybLex/Zeo-MS2

Citations & References (18)

Citations & References
Abstract
Utilization of MHC class I transgenic mice for development of minigene DNA vaccinesencoding multiple HLA-restricted CTL epitopes.
Authors:Ishioka GY, Fikes J, Hermanson G, Livingston B, Crimi C, Qin M, del Guercio MF, Oseroff C, Dahlberg C, Alexander J, Chesnut RW, Sette A
Journal:J Immunol
PubMed ID:10201910
'We engineered a multiepitope DNA minigene encoding nine dominant HLA-A2.1- and A11-restricted epitopes from the polymerase, envelope, and core proteins of hepatitis B virus and HIV, together with the PADRE (pan-DR epitope) universal Th cell epitope and an endoplasmic reticulum-translocating signal sequence. Immunization of HLA transgenic mice with this construct ... More
Interactions between protein kinase CK2 and Pin1. Evidence for phosphorylation-dependent interactions.
Authors: Messenger Moira M; Saulnier Ronald B; Gilchrist Andrew D; Diamond Phaedra; Gorbsky Gary J; Litchfield David W;
Journal:J Biol Chem
PubMed ID:11940573
'The peptidyl-prolyl isomerase Pin1 interacts in a phosphorylation-dependent manner with several proteins involved in cell cycle events. In this study, we demonstrate that Pin1 interacts with protein kinase CK2, an enzyme that generally exists in tetrameric complexes composed of two catalytic CK2 alpha and/or CK2 alpha'' subunits together with two ... More
Role of CCAA nucleotide repeats in regulation of hemoglobin and hemoglobin-haptoglobin binding protein genes of haemophilus influenzae.
Authors:Ren Z, Jin H, Whitby PW, Morton DJ, Stull TL
Journal:J Bacteriol
PubMed ID:10482534
'Haemophilus influenzae utilizes hemoglobin and hemoglobin-haptoglobin as heme sources. The H. influenzae hemoglobin- and hemoglobin-haptoglobin binding protein genes, hgpA, hgpB, and hgpC, contain lengths of tetrameric CCAA repeats. Using an hgpA-lacZ translational gene fusion, we demonstrate phase-variable expression of lacZ associated with alteration in the length of the CCAA repeat ... More
Regulation of the Bub2/Bfa1 GAP complex by Cdc5 and cell cycle checkpoints.
Authors: Hu F; Wang Y; Liu D; Li Y; Qin J; Elledge S J;
Journal:Cell
PubMed ID:11733064
'During mitosis, a ras-related GTPase (Tem1) binds GTP and activates a signal transduction pathway to allow mitotic exit. During most of the cell cycle, Tem1 function is antagonized by a GTPase-activating protein complex, Bfa1/Bub2. How the Bfa1/Bub2 complex is regulated is not well understood. We find that Polo/Cdc5 kinase acts ... More
Substantially enhanced cloning efficiency of SAGE (Serial Analysis of Gene Expression) byadding a heating step to the original protocol.
Authors:Kenzelmann M, Muhlemann K
Journal:Nucleic Acids Res
PubMed ID:9889294
'The efficiency of the original SAGE (Serial Analysis of Gene Expression) protocol was limited by a small average size of cloned concatemers. We describe a modification of the technique that overcomes this problem. Ligation of ditags yields concatemers of various sizes. Small concatemers may aggregate and migrate with large ones ... More
18 total citations

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