#SERIAL CLONER COHESIVE ENDS PDF#
#SERIAL CLONER COHESIVE ENDS SOFTWARE#

When visualizing DNA bands with a transilluminator, do not use short wavelength UV light of 312 nm or below because the DNA will be severely damaged. In addition to removing unwanted or incompletely digested DNA, this procedure removes enzymes and small molecules. Purify the inserted fragment with a gel.

You can use relatively short digestion times, and for a double digest, you can use a reaction buffer that is suboptimal for one or both enzymes.

For preparation of an inserted fragment, incomplete digestion is not a serious concern because partial recovery of the fragment is acceptable.

It is a good idea to spin briefly after vortexing to ensure that no droplets are left on the side of the tube. Vortex the mixtures thoroughly and repeatedly, and do not allow any droplets of liquid to escape enzyme treatment. With the vector, ensure that the digestion and phosphatase reactions go to completion. Gel purification of the vector is usually unnecessary because phosphatase treatment will inactivate any extra DNA fragments that were generated.

Remove the phosphatase with a spin-column.

After digesting the vector (and blunting, if appropriate), treat with phosphatase: 1 hr at 37☌ for 5′ overhangs, or 1 hr at 50☌ if the DNA has any blunt ends or 3′ overhangs.

If the vector needs to be cut with two enzymes that have different optimal reaction buffers, perform the digests sequentially, with a spin-column purification in between. Use an excess of restriction enzyme (~20 U for 2 μg DNA), and digest for ~4 hr.

Ensure that digestion of the vector goes to completion.

The tricks described below will minimize these effects. This problem has two causes: incomplete digestion of the vector, and re‑ligation of the cut vector with itself. The most common problem with restriction cloning is that the starting vector is recovered after the procedure. Cleaner starting material will yield a better outcome.

When in doubt, purify a DNA fragment with a gel.

For example, it works better to clone a blunt-ended fragment into a blunt vector site, such as a SmaI site, than into a site that has been blunted with Klenow or T4 DNA polymerase.

To maximize efficiency, minimize the number of steps in a procedure.

(There is no need to use a spin-column before purifying a DNA fragment with a gel.) Elute the DNA in 40 – 45 μl of 10 mM Tris (pH 8.5), and then perform the next reaction.

After each enzymatic reaction, purify the DNA with a spin-column.

Start with about 2 μg of DNA when preparing a vector or excising a fragment to be inserted. This approach saves time in the long run.

First and foremost, be careful at each step of a procedure.

A few simple tricks will help to ensure that your cloning goes smoothly. Restriction Cloning Tipsįor many applications, conventional restriction cloning is still the best method. Inframe cloning, Express human erythropoietin gene as fusion proteinĩ.Using a specific example, you will learn to simulate inserting a single fragment into a plasmid vector using restriction and insertion cloning in SnapGene. PCR based cloning, Clone Gene BBK56 into expression vector plasmid bbk56 into pGEX3XĨ. PCR based cloning, Clone GeneY from pTDL11 into cloning plasmid pUC19Ħ. Directional cloning with compatible cohesive endsĥ. Directional cloning, using reverse complement functionĤ. Directional cloning, Sub-clone human erythropoietin gene from plasmid pTDL14 into expression vector pGEX-5X3ģ. Sub-clone “gene X' from vector pTDL10 into cloning plasmid pBR322Ģ. Any cloning enthusiast is welcome to try it!ġ. Researchers can store/organize entire cloning experiments, strategies and new molecules in computer.ģ. Researchers of the lab will get skilled in developing cloning strategies using software. Research laboratories, which often perform cloning experiments. Students will also become skilled in developing cloning strategies using software.Ģ. They will find it exciting to perform in silico (in computer) experiments and put their knowledge to test. Students who have read gene transcription/ translation from textbooks. Who can use “Computer based exercises in Molecular Cloning Strategies?'ġ.

#SERIAL CLONER COHESIVE ENDS SOFTWARE#

A detailed video answer that takes you step-by-step and click-by-click through the entire solution on SerialCloner software (One can download SerialCloner free from GenBank files of molecules discussed in the questionģ.

#SERIAL CLONER COHESIVE ENDS PDF#

A PDF that describes the questions and explains answer as textĢ. It's a set of nine exercises where you can simulate cloning and gene expression experiments in computer.

Serial cloner cohesive ends