Cloning Protocol
PCR
There are two types of PCR polymerases used in the lab, TAQ and Phusion. Phusion is more accurate, having fewer mutations or errors in PCR. TAQ will almost always give a good product, and is used when accuracy is not an issue. TAQ has an error rate of about 1 bp change per Kb. The protocols for these are very similar
Phusion
50 ul reaction
1. ddH20 – 35.5 ul
2. 5X Phusion buffer— 10 ul
3. 10 mM dNTP’s— 1 ul
4. Primers (50 uM stock)—1 ul each for 2 ul total
5. Template DNA—1 ul
6. Phusion Polymerase— .5ul
====a.==== The polymerase is VERY expensive so make sure that you only get .5 uL per reaction.
====b.==== Make sure you just get out the polymerase after the other stuff is already mixed in to ensure the proper pH and to make sure that the polymerase stays cold. As with all enzymes, ALWAYS use the blue freezer boxes to keep it cold at your bench.
TAQ
50 ul reaction
1. ddH20— 41 ul 2. Standard 10X reaction Buffer— 5ul 3. 10 mM dNTP—1 ul 4. Primers—1ul each for 2 ul total (1ul forward and 1ul reverse) 5. DNA template—1ul of liquid or 1 colony 6. 1x TAQ DNA polymerase—1 ul
====d.==== After completing the reaction mix you will need to place it into the PCR machine. Sign into your profile and then select the program that correlates to the PCR reaction you will be running. Press start and then select tubes, or plates. It will usually take anywhere from 2-3 hours for shorter products. For the extension time, TAQ requires 1 min per kb of product and Phusion requires 2 min per kb of product. RECORD the PCR conditions, the primers, template and program used in your notebook.
DNA Electrophoresis
After completing the PCR you will need to run it out on gel. The purpose is to determine if the product is correct by visualizing the size of your PCR product (it should be the size you wanted for your gene of interest).
a. To make the gel you will need 75 ml of TAE and .75 grams of agarose (regular agarose, not low melt).
b. Put it into the microwave for about 90 seconds or until the agarose is completely dissolved.
c. Add 7.5 ul of ethidium bromide, make sure you wear gloves because it is a carcinogen.
d. After it is has cooled briefly, pour the liquid onto the gel bed and let it cool. It should only take about 30 minutes for it to cool.
e. You will need to have a ladder in one lane and then place your PCR products in the others. You will need a ladder in each row to be able to determine the size of your PCR products. We have 6 ul ladder which means you will just need 6 ul of the ladder. We have 10X dye which you will add to the PCR product to make it heavy enough to lad into the gel (the dye contains glycerol) . We typically run 10 uL of PCR reaction to confirm there is a product . Do this by adding 5 uL of 10X dye to 50 ul of PCR , then load only 10 uL of this mix.
f. Move the gel into the proper orientation in the gel box, cover your gel with 1X TAE buffer and load all your products into the gel.
g. Put the lid on and make sure that the negatives and positives are in the correct places (Red to red, black to black. DNA is negatively charged so the DNA will run toward the red probe) . Turn on the power supply and to run around 150-175 volts. It will take about 30-45 minutes to complete.
h. Turn it off and then the check it on the alphaimager. Print off the results and log in your notebook.
PCR Qiagen Purification Kit
a. This step will help to purify the DNA product away form the buffer and enzymes present in the PCR mix. You can follow the procedure in the Qiagen manual.
Restriction Digest
50 ul reaction
The purpose of this restriction digest is to cut the plasmid open and so that the gene of interest can be inserted into the place where the plasmid will be cut. You will need to have the proper restriction enzymes picked out so that they cut the plasmid in the proper place. There will be two different reactions. One will be with the Purified PCR product and the other will be the plasmid that the gene will be inserted into.
Purified PCR reaction
1. 5 ul NEB buffer (it depends on the R.E. used) 2. .5 ul 100X BSA 3. 40 ul PCR product 4. 1.5 ul Rectricion enzymes (total of 5 ul, 2.5 of each one)
Plasmid
1. 30 ul ddH20 2. 5 ul 10X NEB buffer 3. .5 ul 100X BSA 4. 10 ul of plasmid (3ug) 5. 1.5 ul Restriction enzymes (total of 3 ul, 1.5 ul of each one) ==b.== After all of these are mixed together they must be placed in the 37º bath for at least two hours or overnight.
Low-melt agarose gel
==a.== It is basically the same as the other gel except for 3 things, there is low-melt agarose gel powder that must be used, and it will only need to be in the microwave about 50 seconds or until completely dissolved. Second, it polymerizes slowly so put it in the fridge to solidify. Third, when running the gel, run at 70-90 Volts (do not go over this voltage or else it will melt). It will take about 1 to 1.5 hours to run.
==b.== After it is done running you will use the portable UV lamp and you will extract the products right out of the gel (get help if you do not have a lot of practice at this). Label each tube and place the products into a 1.5 ml eppendorf tube. Be sure to cut out as small of a slice as possible to avoid agarose which inhibits the ligation. It is okay to cut away some of the DNA, you just want to get the DNA to be as concentrated as possible. Also cut out a small slice from an area of the gel that has no DNA as a control for the insert.
Ligation
a.
The purpose of this is to combine the digested plasmid with the gene of interest and then ligate the gene of interest into the plasmid. You will need to set up at least two reations. The plasmid only with insert control and the plasmid with your gene of insert as the insert. To be able to use the DNA you will need to melt the gel at 65 degrees Celsius for about 5 minutes. While it is melting, make a master mix that has everything but the agarose slices and distribute into eppendorf tubes. Add the agarose slices quickly after they melt (before they resolidify). NOTE: you will need a tube for each insert, plus one for the plasmid only control.
Plasmid only
1. 3 ul of plasmid slice
2. 3 ul of insert control slice
3. 6.5 ul of ddH20
4. 1.5 ul 10X ligase buffer
5. 1 ul T4 DNA ligase
Plasmid plus insert
1. 3ul of Vector slice
2. 3ul of insert
3. 6.5 ul of ddH20
4. 1.5 ul 10X ligase buffer
5. 1ul T4 DNA ligase
b.
These reactions will need to incubate at room temperature for at least an hour.
Transformation
a.
Now, if all has gone well, the gene of interest will be in the plasmid and all that will be left to do is to put the plasmid into E. coli (or use other competent cells). The E. coli we use is DH5alpha.
1. Get a cooler and put ice into it (Make sure you have 42 degree water bath set up for a later step). You will also need to get the plates out with the proper selection marker.
2. Get the DH5alpha cells out of the -80 degree Celsius freezer and put them directly into the ice. You will need about 25-30 ul for every reaction you have.
3. They will need to thaw on the ice for a few minutes.
4. After they have cooled you will need to put 2 ul of the ligation mix and add it to about 25-30 ul of competent cells. Vortex them and put them back in the ice for 2-30 minutes
5. Heat shock them by putting them in the 42 degree water bath for 1 minute.
6. Put them back on ice for 2-5 minutes
7. Add .5 ml of LB to the competent cells for 30 minutes.
8. Put all of the mixture into onto the plates and then use the large beads to put the cells all across the plate.
9. Incubate them at 37 degrees Celsius
Checking the clones
a.
After the 24 hour incubation period, if the cloning worked then you should see individual colonies that hopefully contain the plasmid with the gene of interest in the plasmid. This is what you will need to check. (it is important that you do not incubate them over 24 hours because doing this creates “satellite colonies” which the surrounding cells have used up all of the Amp and thus do not have the plasmid in them but can survive because there is no Amp in that part of the plate).
b.
There are two ways to check your clones one is by doing a dirty plasmid prep and the other is to check it using PCR
Checking with PCR
Checking with PCR (you will need to have LB+selction plates out to plate each colony that you are testing. If one of them has the gene of insert in it then you will need to have something to go back to use it more.
1.
TAQ (on the PCR matching you will need to add a 95 degree Celsius for two minutes at the beginning, this will break open the cells)
=====a.===== For a 20 ul reaction
i. ddH20— 16.5 uL
ii. Standard 10X reaction buffer—2uL
iii. Primers—.5 uL each for for a total of 1 uL( .5ul Forward and .5 uL reverse)
iv. DNA template—1 colony (use colony from the transformation plate)
v. 1X TAQ DNA polymerase—.5 uL
Checking using a dirty plasmid Prep
1. After the transformation is done you will need to streak for single colonies. To do this you will need to get LB+ selection plates out. You will pick one and only colony off the transformation plate and then the plate and streak them onto the new plate. Incubate them for 24 hours at 37 degrees Celsius.
2. Now you will need to make overnights of a single colony off the plates you just streaked the day before. You will need to get test tubes out and put about 2mL of LB+selction in them. Label your tubes.
3. Once you have the tubes you ready, you will take a pipet tip in your hand and you will take one individual colony off and drop it into the test tube. For a control leave a test tube with just LB+selction (no colony). After all of the samples are ready you put them into the 37 degree shaker and place the plates into the 37 degree incubator for 24 hours.
4. After 24 hours you will take the test tubes out of 37 degree shaker. Get 1.5 mL eppendorf tubes out for each sample. You will pipet out about 1-1.5 mL from the test tube and put it into the eppendorf tube.
5. Spin down the eppendorf tubes at 13,000 rpm for 2 minutes. After the spin is done decant all of the LB out. It will be on the top. The cells will be on the bottom. You just want the pellet (cells).
6. Place them in the freezer for about an hour or more. This will help them to be able to break open more easily.
7. Remove them from the freezer and then make the tens solution. (Make sure that you use 10M NaOH to dilute it down to 1M NaOH. )
8. Here is the mix that you will need to make in order to make the plasmid prep per sample.
a. 300 uL TENS
b. 3 uL NaOH
c. .6 uL RNAaseA
i. for example if you have 10 reactions to do then you will need to mix
1. 3000 uL of Tens
2. 30 uL of NaOH
3. 6 uL of RNAaseA
9. Put 300 uL of TENS into each eppendorf tube with you pellet. You want your pellet mixed in well with TENS so it is easiest if you pipet up and down to mix it all together.
10. Pipet 150 uL of NaAc (sodium acetate) pH. 5.3 into each eppendorf tube. NO NOT VORTEX it will shatter the plasmid. Invert the tube up and down 4-6 times to mix it all together.
11. Place in centrifuge, 13,000 rpm for about 4 minutes.
12. The plasmid will not be in the supernatant, you will want to get new eppendorf tubes and pipet out the supernatant and put it into the new tubes. Throw away the debris.
13. Put 900 uL of cold 95% ETOH into each tube.
14. Centrifuge them for 2 minutes at 13,000 rpm.
15. Decant the ETOH
16. Add 1 mL of 70% ETOH and put them back in the centrifuge with the same settings as before.
17. Decant ETOH and repeat step 16.
18. After the second spin make sure you decant all of the ETOH. Let the eppendorf tubes air dry for about 5 minutes or until all the ETOH has evaporated.
19. You should be left with a white pellet.
20. Add 50 uL of EB buffer from the qiagen kit.
Restriction Digest
1. Mix plasmid in with the EB buffer.
2. Put 10 uL of plasmid into eppendorf tubes.
3. Make a master mix with your restrction enzymes. You will only use a 10 uL of mix per tube.
a. 8 uL of ddH20
b. 2 uL of NEB buffer
c. .2 BSA
d. .2 Restrction enzyme A
e. .2 Restriction enzyme B
4. Put the mix of 10 uL of restriction enzyme mix, with 10uL of plasmid into the 37degree water bath for at least 1 hour.
5. Make a gel and run the 20 uL reaction on the gel. You should be able to see the size of the plasmid along with the insert.
6. If the insert looks like it is the right size. Follow the next protocol.
Sequencing gene of interest
1. Purify your plasmid by making a 5 mL overnight. Take a colony from the plate and place it in the test tube.
2. Spin the cells down and use the qiagen kit to purify your plasmid.
3. Dr. Grose will need to place an order for you on the DNA sequencing center website.
4. The mix you will need to make is
a. 8 uL of ddH20
b. 2 uL of (1/20 dilution of primers) Talk to Dr. Grose about which primers you need to use. They are not the same as the ones that you did your orginal PCR product with.
5. Take to room 690 in the widsoe for sequencing. Ask someone who has gone before for the exact details of what to do there.
Blunting After Restriction Digest
(Preheat a block incubator to 75C first) a. After the restriction digest perform a Qiagen PCR clean-up kit to each sample. b. Next make a 1:10 dilution of 10mM dNTPs c. Then to each sample add the following to your DNA/plasmids: 1. 1ul of 1:10 dilution of 10mM dNTPs 2. 10ul of 5X Phusion Buffer 3. 3ul of Phusion Polymerase d. Incubate the samples in a 37C water-bath for exactly 10 minutes, DO NOT GO
LONGER!!!
e. Immediately place the samples in the 75C block incubator for 10 minutes (this
inactivates Phusion Polymerase)
f. Perform another Qiagen PCR clean-up kit to each sample g. Perfrom CIP (Phosphotase) treatment to the plasmids only (do not do this to the
DNA insert) to keep them from re-ligating together. This is accomplished by doing the following:
1. Add 5ul of NEBuffer 3 to each plasmid sample 2. Add 1ul of CIPhosphotase 3. Incubate all samples in 37C water-bath for 1 hour 4. Immediately place sample in 75C block incubator for 10 minutes (this inactivates CIP) h. Finally run your samples on low-melt agarose gel and isolate your fragments of
interest and perform ligation as described in above methods.
