Alkaline phosphatases are routinely used to reduce the background from empty, religated vectors during cloning of DNA fragments, since dephosphorylated DNA termini cannot be ligated by DNA ligase. The phosphatase treatment will effectively reduce the background of “empty” clones by >95%.
Why do we use alkaline phosphatase to prevent vector regeneration?
Thus the phosphatase treatment of the vector molecules increases the proportion of hybrid DNA molecules in the population of transformed cells, and also prevents the formation of oligomeric forms of the vector. Dale, J.W. and Greenaway, P.J. (1985) “The use of alkaline phosphatase to prevent vector regeneration” Methods Mol. Biol. 2 :231-236.
What is the role of a phosphatase in vector ligation and transformation?
If a vector is linearized by a single restriction enzyme, or has been cut with two enzymes with compatible ends, use of a phosphatase, such as Quick CIP, to remove the 5´ phosphate reduces the occurrence of vector re-closure by intramolecular ligation. Decreased re-circularization reduces the background during subsequent transformation.
How can I prevent self-ligation of the prepared cloning vector?
If the ends of the prepared cloning vector are identical (e.g., cut with a single restriction enzyme), treat the vector with a phosphatase like TSAP Thermosensitive Alkaline Phosphatase (Cat.# M9910) to remove the phosphate groups from the 5′ ends and prevent self-ligation of the vector.
What is dephosphorylation and how is it used in cloning?
Dephosphorylation is a common step in traditional cloning workflows to ensure that the vector does not re-circularize during ligation. If a vector is linearized by a single restriction enzyme, or has been cut with two enzymes with compatible ends, use of a phosphatase, such as Quick CIP, to remove the 5´ phosphate reduces...
What is phosphatase treatment for?
To minimize self-ligated vector in your transformation, treat your linearized vector with a phosphatase to remove the 5' phosphates necessary for ligation. This should improve the percentage of colonies with inserts.
Why do I need to dephosphorylate my vector?
Dephosphorylation of the vector is done if you want to insert a fragment into a vector and need to prevent re-ligation of the vector without any insert. So you dephosphorylate after you have cut the vector, then purify the cut vector before adding the insert.
What is the role of alkaline phosphatase in creating recombinant vectors and why is it important?
Alkaline phosphatase removes 5' phosphate groups from vector so that prevents self-ligation of the vector and facilitates the ligation of other DNA fragments into the vector.
Why is the cleaved plasmid DNA often treated with alkaline phosphatase prior to the ligation step?
For gene-cloning experiments, why is the cleaved plasmid DNA often treated w/ alkaline phosphatase prior to ligation step? ligase can join the plasmid DNA that has been treatment with alkaline phosphatase and the target DNa and form two phosphoeister bonds.
What is CIP treatment?
CIP treatment is done to phosphatase the vector used for plasmid ligations. This is done to reduce the self ligation of a vector digested with enzyme(s) creating compatible sticky ends and hence enhancing the Signal/Noise ratio of transformations.
Is phosphorylation necessary for ligation?
Since the vector has been dephosphorylated, and ligation requires the presence of a 5'-phosphate, the insert must be phosphorylated.
What is the role of alkaline phosphatase in genetic engineering?
Description: Alkaline Phosphatase is suitable for removal of terminal monoesterified phosphates from deoxyribo-oligonucleotides. Used for the removal of single phosphate groups from 5´-ends of linear vectors to prevent re-circularization during cloning or to dephosphorylate DNA prior to kinase labelling protocols.
How are ends of DNA modified by alkaline phosphatase?
There are two immense uses for alkaline phosphatase in DNA modification: 1. Removal of 5' phosphates from plasmid and bacteriophage vectors by restriction enzyme. In subsequent ligation reactions, this incision prevents self-ligation to facilitate the ligation of other DNA fragments into the vector (e.g. subcloning).
What is alkaline phosphatase enzyme activity?
Alkaline phosphatases are widely distributed enzymes (e.g., liver, bile ducts, intestine, bone, kidney, placenta, and leukocytes) that catalyze the release of orthophosphate from ester substrates at an alkaline pH. The normal activity level in adult serum is highly dependent on the measurement method, age, and sex.
How is phosphatase related to the ligation reaction?
How is phosphatase related to the ligation reactions? Explanation: Phosphatses are used to stop unwanted ligation. It is so because if phosphatases are present, phosphate would be removed from the ends and it would further block the ligation. It is so because the phosphate group is necessary for ligation to take place.
What is the purpose of ligation?
Ligation of DNA is a critical step in many modern molecular biology workflows. The sealing of nicks between adjacent residues of a single-strand break on a double-strand substrate and the joining of double-strand breaks are enzymatically catalyzed by DNA ligases.
Why is it important to use restriction enzymes with sticky ends for cloning?
Restriction enzymes cut double-stranded DNA in half. Depending on the restriction enzyme, the cut can result in either a sticky end or a blunt end. Sticky ends are more useful in molecular cloning because they ensure that the human DNA fragment is inserted into the plasmid in the right direction.
What is dephosphorylation in cloning?
Dephosphorylation is a common step in traditional cloning workflows to ensure that the vector does not re-circularize during ligation. If a vector is linearized by a single restriction enzyme, or has been cut with two enzymes with compatible ends, use of a phosphatase, such as Quick CIP, to remove the 5´ phosphate reduces the occurrence ...
What is the mechanism of dephosphorylation?
The Mechanism of Dephosphorylation. Dephosphorylation is the process by which phosphate groups are removed from a molecule by a phosphatase. Removal of phosphate groups from a DNA fragment can prevent ligation. Learn more about dephosphorylation and phosphatases.
What is dephosphorylation in cloning?
Dephosphorylation is a common step in traditional cloning workflows to ensure that the vector does not re-circularize during ligation. If a vector is linearized by a single restriction enzyme, or has been cut with two enzymes with compatible ends, use of a phosphatase, such as Quick CIP, to remove the 5´ phosphate reduces the occurrence ...
What is the mechanism of dephosphorylation?
The Mechanism of Dephosphorylation. Dephosphorylation is the process by which phosphate groups are removed from a molecule by a phosphatase. Removal of phosphate groups from a DNA fragment can prevent ligation. Learn more about dephosphorylation and phosphatases.
What enzymes are used to fill 5′-protruding ends of a cloning vector?
Both Klenow (DNA Polymerase I Large Fragment) and T4 DNA Polymerase can be used to fill 5′-protruding ends with deoxynucleotide triphosphates (dNTPs).
What enzymes leave a 3′ overhang?
For those enzymes that leave a 3′ overhang, T4 DNA Polymerase has a 3’→5′ exonuclease activity that will, in the presence of excess dNTPs, convert a 3′-protruding end to a blunt end.
Can a linear vector be cut and dephosphorylated?
For linear vectors with unique 5′ ends, dephosphorylation is not necessary. The dephosphorylation reaction can be performed directly in restriction enzyme buffer so the vector can be cut and dephosphorylated at the same time.
Can restriction enzymes be used for PCR?
While T-vector cloning is commonly used for PCR-ampl ified inserts, restriction enzymes still have their uses. For example, you can ensure directional cloning if you digest a vector with the same two enzymes like BamHI and EcoRI that are used to digest your insert.
Can cloning be used to modify the end of vectors?
However, there are other cloning techniques that can be used to modify the end of vectors and inserts after restriction enzyme digestion and prior to ligation.
How to isolate insert and vector?
Isolate your insert and vector by gel purification: Run your digested DNA on an agarose gel and conduct a gel purification to isolate the DNA. When running a gel for purification purposes it is important to have nice crisp bands and to have space to cut out the bands.
How much DNA should be fused in a ligation reaction?
Conduct a DNA Ligation to fuse your insert to your recipient plasmid. We recommend around 100ng of total DNA in a standard ligation reaction. You ideally want a recipient plasmid to insert ratio of approximately 1:3.
What is subcloning by restriction digest?
Subcloning by restriction digest is a commonly used lab technique. For the purposes of this tutorial we will discuss how to move a cDNA from one plasmid to another. However, the same technique can be used to move promoters, selectable markers, or any other DNA element between plasmids.
How long does it take for plasmids to digest?
It is also critical that as much of the recipient plasmid as possible be cut with both enzymes, and therefore it is important that the digest go at least 4 hours and as long as overnight. If you are going to use only one restriction enzyme, or enzymes that have compatible overhangs or no overhangs after digestion, ...
How many colonies do you need to pick for DNA purification?
Finally, you will need to pick individual bacterial colonies and check them for successful ligations. Pick 3-10 colonies depending on the number of background colonies on your control plate (the more background, the more colonies you will need to pick) and grow overnight cultures for DNA purification.
Can you use a single enzyme?
It is also possible to use a single enzyme, but this will require phosphatase treatment of your recipient plasmid as well as a specifically designed test digest later to verify that the insert was cloned in the correct orientation. If you cannot find enzymes that meet these criteria, do not fear. You have other options, such as: ...
Can you use PCR to flank oligos?
You have other options, such as: Adding desired restriction sites to flank your insert : You can use PCR Based Cloning and add restriction sites to the ends of your oligos. This will allow you to produce a version of your insert flanked by restriction sites compatible with the recipient plasmid's MCS.
What is the protocol for insert and vector DNA ligation?
Protocol: Standard Insert + Vector DNA Ligation. Before setting up the ligation reaction itself, it is important to determine the amount of cut insert and vector to use for the ligation reaction. The volume of vector DNA and insert DNA used in the ligation will vary depending on the size of each and their concentration.
What is the final step in the construction of a recombinant plasmid?
The final step in the construction of a recombinant plasmid is connecting the insert DNA (gene or fragment of interest) into a compatibly digested vector backbone. This is accomplished by covalently connecting the sugar backbone of the two DNA fragments. This reaction, called ligation, is performed by the T4 DNA ligase enzyme.
Why is the sticky end of a vector more likely to ligate to itself?
This ensures that the insert will be added in the correct orientation and prevents the vector from ligating to itself during the ligation process. If the sticky ends on either side of the vector are compatible with each other, the vector is much more likely to ligate to itself rather than to the desired insert.
What is the role of DNA ligase in bacterial cells?
The DNA ligase catalyzes the formation of covalent phosphodiester linkages, which permanently join the nucleo tides together. After ligation, the insert DNA is physically attached to the backbone and the complete plasmid can be transformed into bacterial cells for propagation.
Do vector alone ligase control?
Do controls: When doing ligations you should ALWAYS do a vector alone + ligase control. This will allow you to verify that the vector was completely digested and if phosphatase treated, that the phosphatase treatment worked. This control should, in principle, be free of colonies, but the reality is that it will have some amount of background. What you want to see is that your vector + insert ligation has many more colonies than your vector alone ligation.