8 Plasmid Cloning Procedures You Need to Know

Transcription

1 8 Plasmid Cloning Procedures You Need to Know And Some Tips for Mastering Them Free for Personal Use Try it Now

2 8 Plasmid Cloning Procedures You Need to Know (And Some Tips for Mastering Them) 2012 BioData Ltd. All rights reserved. Content may be copied for personal use. It may not be copied or distributed for other purposes without prior written consent from BioData Ltd. Copies may be acquired by downloading from Thank you for downloading 8 Plasmid Cloning Procedures You Need to Know (And Some Tips for Mastering Them) from Labguru.com. Labguru creates digital tools for increasing the produc2vity of academic and industrial laboratories. Our web based so;ware is currently used by thousands of researchers in hundreds of labs worldwide. It is available FREE for personal use. To get started, check out. Free for Personal Use Try it Now

3 8 Plasmid Cloning Procedures You Need to Know And Some Tips for Mastering Them The basic procedure performed by any molecular biologist is, naturally, cloning. You may wish to clone a DNA fragment into a plasmid for many purposes, such as protein expression and purification, gene knock out / silencing / overexpression, reporter gene fusion to a gene of interest, and many more. Whether you plan on doing one thing or another with the plasmid you cloned, when it comes to the cloning procedure you will need to perform the same steps, which are the ABC s of molecular biology. However simple it may seem, when you don t have enough experience in cloning plasmids, you can find yourself struggling endlessly, going over the same protocol over and over again for weeks and even months, gaining no success or insights as to what went wrong. I ve seen so many frustrated graduate students and postdocs walking around the corridors of research buildings, knocking on neighboring lab doors looking desperately for help and advice. I certainly do not claim to be a cloning guru, on the contrary. I ve had my fair share of nightmare-haunted nights, full of dreams featuring linear vectors unwilling to circulate, and images of me in front of the PhD committee having nothing to discuss. It took me quite a while (and many frustrations and disappointments) to realize that before jumping into the icy cold water of molecular biology protocols, I need to thoroughly read, learn and consult experts. It s as simple as that: the more you know about the cloning protocols, understand the rationale behind each step, and the function of each component used, the faster and easier you ll cross the bridge from preparing the molecular tools for your research to the actual science making. We therefore prepared here for you, the molecular biologist, a step-by-step protocol for cloning plasmids. For each step you ll find some useful suggestions and recommendations. The procedures discussed in this protocol include: Polymerase chain reaction Ligation Bacterial transformation Plasmid DNA isolation And more You are invited to download and use the protocol as a basic guide to your cloning. Good luck in your cloning! Free for Personal Use Try it Now 3

4 1) Plan your cloning strategy and arrange your data Begin with data collecting - make sure you have a very clear plan of What your insert DNA fragment is going to be Which plasmid vector you ll use as recipient of your insert Their complete sequences, and What restriction enzyme you ll use at each end of the insert. Based on my experience, good planning leads to fast and successful cloning. You do not need to reinvent the wheel. Most probably, someone in your lab has already cloned the same insert you are about to. If your lab is organized this should not be too difficult or time consuming. Simply go to whatever management system your lab uses, and search for possible origins for your insert. Make a list of all the available vectors bearing that insert, and their locations in stock boxes. Prior to getting back to the bench don t forget to consult the person who cloned the vector you re about to use, for specific advice on practical aspects of working with this vector. The experience of your lab-mates is invaluable! Free for Personal Use Try it Now 4

5 2) Polymerase Chain Reaction PCR The best way for obtaining large amounts of your desired insert is, naturally, using PCR. a) Start your PCR planning by carefully designing your primers. Correct primer design is key factor for a successful PCR that yields the desired product. Don t forget to include the relevant restriction site on both primer edges fitting the orientation of cloning into the recipient vector. If possible, use a different restriction enzyme at each edge, but try choosing two enzymes that can digest in the same buffer, to save time by performing a double digest. When you design your primers pay attention to their sequence at the 5 end; you need to add a few extra nucleotides that precede the restriction site you planted at the 5 end in order to reach higher percentages of PCR product digestion. Determine those extra nucleotides according to the manufacturer's recommendation for each enzyme. Once you have finished editing the primers sequences, it is very important that you also make sure there are no secondary structures, or primer dimers, and that the Tm of both primers is similar. Don't forget that, since in most cases, the 5 end of your primer is not complementary to the sequence amplified due to the restriction site planted, the Tm calculation should not include the non-complementary nucleotides. There are many primer calculation tools available on the Internet for free. As someone who performed PCRs galore, I advise you, do not skip this test. You ll find that if the designed primers were not optimized for the reaction intended for them, you end up wasting precious time, materials and money, but you do not get your desired PCR product. b) Don t use just any Taq polymerase present in the lab. If your PCR is meant to amplify a product that will serve for expression, it is necessary to use a proofreading enzyme. Also, verify that the Taq you use is capable of efficiently amplifying the length of your product, some Taq polymerases cannot synthesize more than 1-2 Kb. (Continued next page ) Free for Personal Use Try it Now 5

6 2) Polymerase Chain Reaction PCR ( Continued) c) Include as many controls as possible in the reaction. Preparing a few more reactions will eventually save you a lot of time and effort when, trying to decipher your results, you ll find yourself straining to figure out what went wrong, why you see no bands in the gel, or what are the extra mysterious bands you do see that are of unexpected length. I recommend for each PCR a no template reaction for each primer pair, and at least one amplification of a positive control product, to check that your PCR mix works well. d) Arrange and organize ahead all the ingredients for your reaction, and mark your tubes so that it is clear what is to be added into each. Make a list of the ingredients, calculate the volumes to be added of each, and make time for preparing and running the reaction. Programme the PCR cycler prior to mixing all the components of the reaction. It may seem as if you can put it together all in 5 minutes in between experiments, but, believe me, if you are not focused on the preparation and mixing of all the components in the correct volumes and order, you ll soon enough be asking yourself questions such as: did I add the dntps to the mix tube? was that primer diluted already? did I add the positive control template? why do I have 100 microliters left in my mix tube even though all sample tubes seem full? e) After the reaction is done, load a small volume of each sample on agarose gel and check if it went well. Don t forget to document! You might want to repeat this protocol or change it a bit. If you have an annotated picture of the gel next to reaction list and PCR program you won t need to trust your memory in a few months, when you ll need to retract your steps or repeat them. f) Great! now you have a tube containing your desired insert amplified to a large amount, almost ready for cloning. It is highly recommended to be rid of the PCR template prior to using the insert for downstream enzymatic reactions. You can either perform gel purification using a purification column, or directly apply the reaction onto a PCR product purification column. These columns are commercially available. Free for Personal Use Try it Now 6

7 3) Restriction The next step is to digest both the insert (your PCR product) and the recipient plasmid you chose. a) Carefully read the manuals provided with the restriction enzymes you are about to use! Make sure to use the best fitting buffer for a double digest, and to incubate at the optimal temperature. If a sequential digestion is required, consult the recommendations of the supplier on how to perform it. b) Remember that the definition of one enzyme unit is the number of enzyme molecules that fully digest 1 microgram of DNA in 1 hour, and calculate the number of enzyme units required according to the amount of DNA you put in the digestion reaction. c) No matter how much DNA you put into your reaction it is crucial that the volume of the enzyme added will not exceed 10% of the total reaction volume. This is because of the high glycerol concentration in the enzyme solution. The total glycerol concentration in the reaction should not exceed 5%, or it will inhibit the digestion. d) It is also highly important that you do not over incubate the reactions. Most of the supplied enzymes today will digest a large amount of DNA within 1 hour if placed in optimal buffer and temperature. Check if the enzymes you use posses star activity. Such activity means that if incubated longer than the recommended period, an enzyme might non-specifically digest DNA sequences other then its recognition site. It is therefore important to consult the manufacturer's protocol, and incubate in accordance. e) To shorten the incubation period you can use the fast-digest enzymes supplied by some companies. These are guaranteed to fully digest 1 microgram of DNA in 5 minutes, so you will not need to worry about star activity, but keep in mind these enzymes are costly. Free for Personal Use Try it Now 7

8 4) Ligation Following restriction enzyme digestion of both your insert and the vector you can perform the ligation reaction. a) Insert - Usually, if the restriction enzymes and ligase were all provided by the same manufacturer, you will not even need to purify your insert from the restriction reaction, but make sure you have inactivated the restriction enzymes prior to ligation. Information on how to inactivate each restriction enzyme is provided in the suppliers manual. In case inactivation is impossible, or that the restriction buffer is not suitable for the ligase you re using, you ll need to load the restriction reaction onto a purification column to separate the insert from the restriction enzymes. b) Plasmid - If the digestion has removed a fragment from your plasmid it is crucial that you purify your plasmid to get rid of that fragment, as it will compete your desired insert during ligation. The best way to do that is by purification of the linear from gel. Alternatively, if you re running low on plasmid amounts, you can simply add a third restriction enzyme that does not cut within your plasmid, but has a recognition site in the fragment you wish to be rid of. This will eliminate competition by the fragment excised, and will minimize loss of precious material. Of course, you ll need to inactivate all 3 enzymes used before you go on with the ligation reaction. c) Ligation - The reaction is very simple to combine and includes buffer, plasmid, insert and the ligase. The most important consideration here is the molar ratio vector : insert. Typically, a ratio of 1:3 works well (3 insert molecules per 1 vector molecule). You ll need to measure the concentration of the vector and insert and combine them accordingly. Always read carefully the manufacturer s supplied manual of your ligation mix prior to performing the reaction, to know the exact incubation conditions. d) Control - If you have enough material it is highly recommended to perform a self ligation reaction of your vector only with no insert added. This will help you get an idea of the efficiency of insert-ligation versus plasmid re-circularization before you start analyzing bacterial colonies and find out a few days later you have no insert in the plasmids you obtained. (Continued next page ) Free for Personal Use Try it Now 8

9 4) Ligation ( Continued) e) To make sure your ligation worked prior to moving on with the bacterial transformation it is recommended to load a small volume of the reaction on a gel next to the linear plasmid mixed with the insert, but with no ligase added. Positive result for ligation should be displayed as bands of various lengths on the gel, indicating that your ligase is intact, and managed to connect the DNA fragments in your ligation solution with one another; each band on the gel containing a different number of ligated fragments. f) Finally, if you have unsuccessfully performed the same ligation over and over again, try dephosphorylating your vector before moving on to ligation in order to eliminate the possibility of self-ligation. Another tip for increasing ligation efficiency is to heat inactivate the ligase prior to transforming the ligated plasmid into bacteria. Free for Personal Use Try it Now 9

10 5) Bacterial Transformation After performing the ligation reaction, you need to deliver the plasmid DNA into host bacterial cells for massive production. At this point you need to consider two factors: 1. Which strain of bacteria to use, and 2. What type of transformation technique to conduct. Bacteria: Note that there are many bacterial strains that have been engineered for various aims. You should therefore choose those for cloning and maintenance purposes. The strains DH5-alpha, XL-1 blue or JM109 will serve you well (you can view a complete list of E. coli strains here). It is critical to know this before hand, as transformation requires the preparation of bacterial cells before the introduction of your ligation (see below). Technique: You can use electroporation, which is known to be more efficient by about three orders of magnitude in comparison to the heat shock transformation method, but requires dedicated electroporator and special corvettes. The heat shock method only requires a wet or dry heater block set to 42 degrees Celsius, which is commonly found in molecular biology labs. Take into consideration that each technique requires a different preparation (or purchase) of your choice competent strain. Opt for the electroporation technique if you can, especially if you routinely find your LB plate empty of colonies. Following transformation - incubate your plates overnight at a 37 O C incubator. Free for Personal Use Try it Now 10

11 6) Clone Verification Good morning! Open the incubator s door, and look at your plates...many bacterial colonies? That s great! Most probably this means your ligation worked, unless you have a similar number of colonies on your self ligation plate. If you don t, the next step for you is to look for positive colonies, containing your plasmid. You don t need to check more than 15 colonies if on your self ligation plate you have much less colonies than on your ligation plate. A common way of performing this test is colony PCR, for amplification of your insert directly from lysed bacterial cells containing the plasmid you cloned it into. In this procedure you perform a regular PCR using bacterial colonies as templates. Include in the reaction either the primers used to amplify the insert, or primers complementary to plasmid sequences on both ends of the insert: First, prepare a Petri dish marked with numbers 1-15 (for each colony you ll check). The plate should contain the same medium used for yesterday s plating - don t forget the antibiotics. Next, prepare the PCR mix (that should include all reaction components except for the template) and divide it to pre-marked 1-15 reaction tubes. Mark control tubes as well: positive and negative control. Use a sterile tip to very gently touch one colony from the ligation plate, then, prick with this tip very gently the Petri dish next to the number 1 marked on it. Now, insert the tip into PCR tube marked 1, and slightly mix the solution with the tip. Discard the tip. The bacteria on the tip serve as your template #1 in this reaction. Perform the same procedure with 14 other colonies. Make sure to touch one colony exclusively each time, and don t forget to incubate the Petri dish with the 15 colonies analyzed to allow colony growth. As negative control template use one colony from the self ligation plate, and as positive control template use the plasmid (or genomic DNA) from which you derived your insert. Include other controls as elaborated under the PCR section. Perform the reaction on the thermo cycler pre-programed, but don t forget to perform a first step of 5 minutes at 95 O C. This step is necessary for lysing the bacterial cells, allowing your plasmid (the template) to get into the reaction mix. Once the reaction is done analyze the products on an agarose gel, and mark the colonies from which the insert was successfully amplified - you won t need more than 2 from which to isolate your plasmid. Prepare a small volume liquid culture of the 2 colonies you chose by using colonies that grew on the Petri dish marked Don t forget to add antibiotics to your liquid cultures, and grow them overnight. Free for Personal Use Try it Now 11

12 7) Plasmid DNA Isolation You are one protocol away now from finally having your desired plasmid! Plasmid isolation these days is mostly performed using commercial columns kits of mini/midi/maxi preps. For your purposes, a mini-prep kit will do the trick. You will need to grow your potential positive colonies overnight in 5ml LB (supplemented with appropriate antibiotics) and the next day start the mini-prep protocol according to the manufacturers recommendation. Although these kits are very easy and simple to use you may encounter low yield issues or contaminated plasmid DNA with proteins or genomic DNA. Usually low yield issues are related to either of the following: 1. Low pellet mass - bacteria was not grown to high enough O.D or bacterial lysis due to phage contamination. 2. Inefficient lysis of bacterial cells - this can be caused by inefficient resuspension of bacterial pellet with the hypotonic solution (the first solution). Using mediumspeed centrifuge force (6000g) when centrifuging the overnight bacterial inoculum leads to a more loose pellet which is easier to resuspend. 3. Old washing solution / ethanol was not added to the supplier s washing solution - washing steps between binding and elution of the DNA prep is performed with high concentration of ethanol (>70%). The supplied washing solution should be supplemented with absolute ethanol by the users so make sure you re using a ready-to-use solution. In addition, over time ethanol may evaporate, and thus it s concentration may decrease, leading to partial elution of your bound plasmid. (Continued next page ) Free for Personal Use Try it Now 12

13 7) Plasmid DNA Isolation ( Continued) Protein contamination (reads of 260nm/280nm<1.8) is usually related to insufficient wash steps following the binding of your DNA to the column or to inclusion of particles from the pellet following the cold centrifuge step (Maximum speed for 10 minutes at 4 degrees Celsius). When removing the supernatant from the tube make sure not to touch or include any white particle of the pellet, which contains mainly membrane and protein fraction of the whole bacterial lysate. Salt contamination (reads of 260nm/230nm<1.5) can be the result of precipitation step and might ruin any sequencing effort. Thus, it is recommended to use two wash steps with the wash buffer, making sure to wash even the rim of the spin column. Submit your DNA sample to sequencing with the appropriate primer, either forward or reverse - if your sequence is long (over 800bp) you might need to sequence it both from the forward and from the reverse side, due to the size limitations of the sequencing technique. You should also take into consideration the fact that the first sequenced ~10-30 bp might be hard to interpret, so you might need a different primer. Free for Personal Use Try it Now 13

14 8) Make sure you have cloned the desired plasmid When you receive your sequence file it will most probably be with the extension of.ab1. You can open such a file with Bioedit ( bioedit/bioedit.html) or GENtle ( programs - both are freely available. Once you ve got the sequence opened on your monitor take a look at the chromatogram - if the peaks are flat or not well resolved, your sequencing effort wasn t successful. See interpret.html for a detailed description on how to analyze sequencing results. If you get a good sequence, now s the time to check if whole effort worked. The quickest way to do that is to BLAST it through NCBI s website 1. Scan the top results: if you don t see your gene, than you got a self-ligated vector result. Don t throw away that result yet! You can still search for the ligation point and possibly analyze what might be the cause of the self ligation (maybe only one digestion occurred?). If you do find your gene, don t pop that champagne just yet - scan the sequence well for mutations. Ask a labmate to help you as it is terribly easy to miss a mutation or two. Sequence is 100% identical without any gaps? Congrats, you ve just successfully cloned your gene! Found a mismatch? Don t worry just yet - go back to the sequence chromatogram and identify the position in which this mutation is observed. How does the peak look? Is it an overlap of a neighboring peak or does it look fine? If the peak looks good than you ve got yourself a ligated mutated gene but you should translate your gene and see if it might be a null mutation. Cloning a gene is an arduous effort with lots of challenges, but if you do this for some time you will quickly realize that your efficiency and success rise with time. Don t loose heart in the face of failures - they happen to everyone! Learn from them and clone away again until you get it done! Good luck! 1. PROGRAM=blastn&BLAST_PROGRAMS=megaBlast&PAGE_TYPE=BlastSearch&SHOW_DEFAULTS=on&BLAST_SPEC=&LINK_LOC=blasttab&LAST_PAGE=blastp Free for Personal Use Try it Now 14