For blunt end cloning we use a cut-ligation protocol that works really well. You add the insert + uncut vector backbone together with restriction enzyme, ligase and ligase buffer and cycle the reaction for 20-30 times at 37°C (the temp of your enzyme) for 2 min / 25°C for 5 min, then heat inactivate.
Full Answer
Can I Try my ligation without using CIP?
Are you cutting at a multi cloning site or cutting an insert out of a vector. If it is possible to use a vector that already has an insert, it would be easier to see if you are cutting at both sites and getting complete digestion. You may be able to try your ligation without using CIP if you believe the digestion and purification was good.
What is the use of CIP in DNA extraction?
CIP is useful in many molecular biology applications such as the removal of phosphorylated ends of DNA and RNA for subsequent use in cloning or end-labeling of probes. In cloning, dephosphorylation prevents religation of linearized plasmid DNA.
How can I reduce the risk of self ligation?
Use a phospatase to dephosphorilate the plasmid (preferentialy SAP, you can also use CIP) and USE for example T4 PNK for phosphorilation of the insert, prior to the ligation. That will reduce self ligation to almost 0. The answer depends entirely on your method of cloning.
What is the protocol used for 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.
How do you do a CIP treatment?
Procedure: Dephosphorylation of DNADissolve DNA in 1X CIP Buffer (0.5µg DNA/10 µL).For 5' overhang DNA add 0.1 units/pmol CIP; for 3' overhang or blunt end DNA add 1 unit/pmol.Incubate 60 minutes at 37 °C.Extract with phenol/chloroform (P3803 or P2069) or gel purify the DNA.*More items...
How does CIP prevent self ligation?
CIP works by removing the phosphate group from the 5′ end of linearised DNA, which means you can use it to dephosphorylate your vector molecule to prevent it from self-ligating and giving you the headache of a high empty vector background after ligation and transformation.
How do you inactivate CIP?
Quick CIP is completely and irreversibly inactivated by heating it at 80°C for 2 minutes, unlike wild type CIP, which is not heat-inactivatable. This makes removal of Quick CIP prior to ligation or end-labeling unnecessary.
How do you use a quick CIP?
The phosphatase can be added directly into the digestion reaction during or after DNA digestion.Add 1 μl of Quick CIP for every 1 pmol of DNA ends (about 1 μg of a 3 kb plasmid) and incubate at 37°C for 10 minutes.Quick CIP is active in all NEB restriction enzyme buffers.More items...•
Can you insert self Ligate?
Hi, I think self ligation of your fragment is possible because it has only one G overhang. You can work without CIP and after ligation and transformation check your fragment size by PCR and Digestion. Between your colonies there will be appropriate colony.
How do you know if ligation is successful?
The presence of high molecular weight molecules after incubation will be indicative of successful ligation. If your insert has ligated to the backbone, then you need to cross check with insert release and see that your insert and vector are released in the same size range as you would know.
What is self ligation?
Takeaway. Self-ligating braces use a built-in mechanism in the bracket to hold the archwire in place. This is in contrast to traditional braces, which use elastic ties or metal wires to secure the archwire. People with self-ligating braces may have shorter orthodontist appointments, easier cleaning, and less discomfort ...
Is dephosphorylation necessary for ligation?
If the vector is dephosphorylated, it is essential to ensure that the insert contain a 5' phosphate to allow ligation to proceed. Each double-strand break requires that one intact phosphodiester bond be created before transformation (and in vivo repair).
How do you turn off alkaline phosphatase?
Inactivation. Alkaline phosphatase can be inactivated by heat: Heat the reaction for 10 min at +65 °C followed by at least one extraction with phenol/chloroform/isoamylalcohol (50 : 48 : 2). AP inactivation by heat depends upon the protein concentration and the divalent ion concentration (Mg2+, Zn2+).
When should I use CIP?
The Mechanism of DNA Phosphorylation CIP is useful in many molecular biology applications such as the removal of phosphorylated ends of DNA and RNA for subsequent use in cloning or end-labeling of probes. In cloning, dephosphorylation prevents religation of linearized plasmid DNA.
How do you dephosphorylate a protein?
To dephosphorylate a protein or DNA, an enzyme or hydrolase that cleaves ester bonds is required. For example, phosphatases remove phosphate groups by hydrolyzing phosphoric acid monoesters into a phosphate ion and a molecule with a free hydroxyl (−OH) group.
Why is alkaline phosphatase treatment necessary in cloning?
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%.
What is the function of CIP?
CIP is useful in many molecular biology applications such as the removal of phosphorylated ends of DNA and RNA for subsequent use in cloning or end-labeling of probes. In cloning, dephosphorylation prevents religation of linearized plasmid DNA. The enzyme acts on 5´ protruding, 5´ recessed and blunt ends. CIP may also be used to degrade unincorporated dNTPs in PCR reactions to prepare templates for DNA sequencing or SNP analysis.
Is CIP enhanced by reducing agents?
CIP is inhibited by metal chelators (e.g. EDTA),inorganic phosphate and phosphate analogs. CIP activity is decreased in the presence of reducing agents (DTT, β-ME).
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.
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 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.
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.
