We value your privacy. Asked 5th May, Rachel Morton. Can I do a double digest when my restriction sites are next to each other? I am digesting a plasmid pcDNA3 with two restriction enzymes and the sites I am using are right next to each other- can I still do a double digest? Does it matter that the restriction sites are next to each other?
Or would I be better off doing two single digests? Thanks for the help! Restriction Digestion. Restriction Enzyme Analysis. Molecular Biology. Basic Molecular Biology Techniques. Most recent answer. Vicken Aknadibossian. University of Florida. This conversation had a blunt end. And i was just making T 4 it. Guess I'l just lie and gaze at it. Popular Answers 1. Robin Dickinson. Aruru Molecular Ltd.Restriction endonucleases recognize specific sequences 4 to 8 base pairs long.
These restriction sites are typically pallindromic - they read identically on both strands. While each restriction enzyme recognizes and cleaves a specific sequence, a given recognition sequence may be recognized by multiple enzymes. Enyzmes which recognize the same sequence are called isoschizomers. Recently these have been further divided into two classes - isoschizomers and neoschizomers - which cleave the DNA at the same position or different positions respectively.
The frequency with which restriction sites occur in a random sequence can be simply calculated if the GC content of the random sequence is known. For each nucleotide position in the restriction site, determine the frequency with which that position is occupied by the appropriate base.
Then multiply the frequencies together to obtain the frequency with which the complete site is observed. Average fragment size and average frequency of restriction enzyme cutting depends on both the length and the GC bias of the recognition site as well as the GC bias of the genome being cut. Biology - Molecular Genetics Restriction Enzymes. Host Restriction. Restriction Enzymes. Naming Restriction Enzymes Restriction enzymes have obscure sounding names which are directly derived from the bacterial strains from which they were first isolated.
Most of the restriction enzymes we purchase commercially have been cloned and are now isolated from engineered E. Several examples of how these enzymes are named are shown below. PstI P rovidencia st uartii 1 st enzyme isolated.
TaqI T hermus aq uaticus 1 st enzyme isolated. Restriction Enzyme Classification Restriction enzymes are classified on the basis of two fundamental properties. First, whether the enzyme cuts at its recognition site, and second whether the restriction and modification activities are associated with the same or different complexes. Type 1: cuts at recognition site restriction and modification activities part of the same complex. Type 2: cuts at recognition site restriction and modification activities part of different complexes.
Type 3 : cuts an arbitrary distance from recognition site restriction and modification activities part of same complex. Most of the enzymes we use in molecular biology are Type 2 enzymes. In the event that we might want to make use of the modification system for instance to protect cDNAs from digestion during library constructionsome type 2 modification enzymes are also commercially available.How would I be able to determine what DNA fragments would be produces?
I need step by step direction of how to look at each plasmid and determine fragment size for each restriction enzyme and if ask for more than restriction enzyme cut?
It depends on how detailed the maps of the enzymes are. If they list the exact positions of the restriction enzymes, you can just calculate the sizes. For example, if you have a 5 kb plasmid, and an enzyme cuts at position, andit will generate a bp fragment -a bp fragment - and a bp fragment.
The size of that last fragment is the size of the plasmid minus the position of cutsoplus the extra bps, because the enzyme did not cut at the origin of replication, which is position 0.
If there are more cuts and enzymes, the situation gets a bit more complex, but I have always enjoyed making these maps. Maps of Two 5 kb plasmids indication recognition sites for 3 different RE enzymes Answer Save. BioLiz Lv 7. Still have questions? Get your answers by asking now.The frequency of cutting in a random DNA sequence for a given restriction enzyme is once per every 4 nwhere n is the number of bases in the restriction enzymes recognition sequence.
Hin dII has a five-base recognition site, so it will cut once per every 4 5or bases. To determine the number of sites, divide the number of bases in the DNA by the average length of the restriction fragments resulting from the given enzyme. The size of l is 48, base pairs, so the number of expected sites are:.
Cutting with Hin dIII will yield fragments of 1, 2, and 2. Cutting with Eco RI will yield fragments of 1. The 1 kb radiolabelled probe is from the second exon in the cDNA.
The radiolabelled probe derived from the wild-type cDNA will only be able to hybridize to the section of genomic DNA that contains the first exon.
Because the length of the Pvu II restriction fragment in the mutant is the same as that of the wild-type, there does not appear to be any deletion between the two PvuII sites. Therefore, the mutation is not the same as that in part d. The mutation here is likely to be a point mutation. There is at least one intron, because the radiolabelled cDNA did not hybridize to the 1.
Other introns may reside in the 2. Two methods that can be used to labed cDNAs are primer extension and nick translation. Primer extension uses an oligonucleotide primer and Klenow fragment to synthesize a labeled DNA strand. The "sticky ends" resulting from digesting with the enzymes are the same, but the actual restriction sites are different. After isolating the plasmid, digest it with both enzymes, purify the 4. You can cut with both enzymes again after ligation — this would ensure that only a plasmid lacking both sites would transform the bacteria.
Walk to a clone that overlaps the end of a translocation or deletion this will show difference in Southern blots from organisms with and without the genetic abnormality. Use a clone from wild-type to pick up a junction fragment in the mutant strain. Use this fragment to pick up a clone at the other end of the chromosomal abnormality from a wild library. Run the mRNA out on a gel and transfer it to a blot this is called Northern blotting.
Using primer extension or nick translation, label isolated genomic DNA and use it as a probe to hybridize to the blot. The probe should hybridize to its corresponding mRNA, and the location of the probe on the blot should tell you the size of the mRNA. To isolate RNA, use Dnase. Use a restriction enzyme, rerun the gel, and add up the band sizes; if more than one DNA, they should add up to greater than the original band.
One method to isolate a D. Another method is to create primers from sequences in the C. Run the mRNA out on a gel, transfer the RNA to a blot, and then hybridize the Northern blot with a labeled probe generated from the gene. Let the wild-type progeny self-mate. If the mutation exhibits incomplete penetrance, then the wild type progeny should yield some mutant progeny.
Only the 4 and 5 kb Bam HI fragments which contain the exons will hybridize on the Northern blots to a 6 kb mRNA and this will only be found in the liver lane. By eliminating the initial 6 kb Hin dIII region, regulatory elements needed for transcription have been omitted. Cosmids are modified plasmids that have the cos cohesive ends of phage lambda and plasmid sequences for replication and drug selection ; they can contain Kb of DNA.
Eco RI. Hin dIII.When cloning by restriction digest and ligation, you use restriction enzymes to cut open a plasmid backbone and insert a linear fragment of DNA insert that has been cut by compatible restriction enzymes. An enzyme, DNA ligase, then covalently binds the plasmid to the new fragment thereby generating a complete, circular plasmid that can be easily maintained in a variety of biological systems. Read on for an in-depth breakdown of how to do perform restriction digests.
Before beginning the restriction digest and ligation process, you should carefully choose your backbone and insert - these both must have compatible cut sites for restriction enzymes that allow your insert to be placed into the backbone in the proper orientation. For instance, if you were cloning a gene into an expression vector, you would want the start of the gene to end up just downstream of the promoter found in the backbone.
Ideally, the backbone will contain a variety of restriction enzyme cut sites restriction sites downstream of the promoter as part of a multiple cloning site MCS. Having multiple sites allows you to easily orient your gene insert with respect to the promoter. You then add ligase to the mixture to covalently link the backbone and insert and, PRESTO, you have a full plasmid ready to be used in your experiments.
Set up restriction digests for your insert or donor plasmid and plasmid backbone. Because you lose some DNA during the gel purification step, it is important to digest plenty of starting material. We recommend 1. It is also critical that as much of the backbone plasmid as possible be cut with both enzymes, and therefore it is important that the digest go until completion.
The time required for complete digestion varies for different enzymes. If you are going to use only one restriction enzyme, or enzymes that have compatible overhangs or no overhangs after digestion, you will need to use a phosphatase to prevent re-circularization of the backbone plasmid see below.
You should treat your digested backbone plasmid with a phosphatase prior to the ligation step or prior to the gel purification step, depending on the phosphatase you choose. You need to isolate your insert and backbone from the enzymes used to digest them as well as any pieces cut out or off of them. An easy way to do this is gel purification. In gel purification, you use a voltage difference across a gel matrix usually agarose to pull your negatively charged DNA through the gel.
As indicated in the figure on the leftyour digested DNA and undigested controls are loaded at the top of the gel in wells positioned toward the cathode - charge. Larger fragments of linearized DNA migrate slower than smaller linearized fragments. You can separate your backbone away from any inserts cut out of it and your new insert from any overhangs cut off of it via their different migration speeds; after running the gel for some duration of time, these differently sized pieces will be in different locations and can be cut out of the gel individually.
There are a variety of ways to visualize the DNA in your gel this table is not inclusive of all gel stains :.Login Register. Login at Biotechnology Forums. Welcome to Biotechnology Forums! Please login using the form on the right Not our member yet? What are you waiting for?! Remember me. Contact: sales biotechnologyforums. Thread Rating: 0 Vote s - 0 Average 1 2 3 4 5. Find number of restriction fragment size in chromosome. Hi all, I am prepare in entrance exam. I need help.
This sequence is found, on average, once every 'X' residues in a chromosome. Find Share.
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ExpertScie Posting Freak. Dear Kanagasundar87, Can you please specify the chromosome in question like its form, source etc.? Is it of human or any other animal etc?AP Biology: Restriction Enzyme Digests on Circular Plasmids
This will help to calculate the same. Or you may share the course type on which these objective questions are being asked. This will give hint to identify the answer.
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Example whenever these are circular pieces of DNA, the fragment number of equals the number of cuts from the restriction enzymes. If plasmid or chromosome is cut at one place by one restriction enzyme then the fragment will be only one. Cutting the circle one will yields only one fragment. If the restriction cuts in two places, it will result in two fragments; with three places, three fragments and so on etc.
In case of linear chromosomes, the situation is slightly different. Cutting a linear chromosome at one place yield two fragments.
How do I calculate the average fragment size that will be cut by a restriction enzyme?
Similarly cutting it at two places results in three fragments etc. So the number of fragments depends upon the type of cut as well as the original base pair of the chromosome in question That is chromosome source and form.
A restriction map of a chromosome will indicate all of the cuts as made by the restriction enzymes. One can count the spaces or gap between cuts to find out the number of fragments that are produced.
DNA gets digested into fragments equal to multiplicity of distance between nucleosomes like, base pairs etc. Hope this will help you to find out the answer. Or please specify the RE type, its source, Chromosome in question to identify the exact answer.Why don't fictional characters say "goodbye" when they hang up a phone? Based on restriction maps of plasmid determine the number of DNA fragments and sizes of the fragments? What evidence does Coutu use to support her claim that improvisation requires resilience.
All Rights Reserved. The material on this site can not be reproduced, distributed, transmitted, cached or otherwise used, except with prior written permission of Multiply. Hottest Questions. Previously Viewed. Unanswered Questions. Wiki User Plasmids are circular pieces of DNA, so the number of fragments equals the number of cuts from the restriction enzymes.
You can easily see this if you start with one restriction enzyme that cuts the plasmid in only one place. Cutting the circle in one place yields you only one fragment. If the restriction cuts in two places, you end up with two fragments; with three places, three fragments, etc. With linear chromosomes, the situation is different.
Cutting a linear chromosome in one place yields two fragments, cutting in two places yields three fragments, etc. So the number of fragments is always one more than the number of cuts. A restriction map of a plasmid will show all of the cuts the restriction enzymes made.
Each cut is labeled with the enzyme that made it. One can count the spaces between cuts to determine the number of fragments that are produced. Restriction maps usually but not always also show the size of each fragment. Related Questions Asked in Genetics What is the simple rule relating the number of fragments to the number of restriction sites presents on the linear DNA molecule? As the DNA fragments results from the action of the restriction enzymes and on the other hand mutations alter the sites where the restriction enzymes react therefore there is difference in number and of length of each fragment from person to person.
Asked in Microbiology, Genetics, Biotechnology What is a multicopy plasmid?