We have been speaking about PCR and how to manipulate this to make all kinds of DNA constructs and mutants.  However, the thermal cycler (aka PCR machine) is almost as useful!  How many things do you do in the lab that require temperature control?  Well, nearly all of them can be done in a thermal cycler.

It's Tips and Tricks Tuesday!

Back before the invention of the thermocycler, PCR was done by transferring reaction tubes from one water bath to another to change the temperature.  Imagine having to sit in front of 3 (or more) water baths transferring the PCR reaction from one, to the next, to the next, back to the first, every minute or so?  This tedium was solved by the programmable thermal cycler that can automatically change the temperature of a specified “block”.  The advent of the heated lid eliminated the need to add an evaporation blocking oil to the reaction, making the reaction easy (and much less messy) to set up and run.

But thermal cyclers aren’t just good for PCR.  We suggest using them for everything that requires controlled temperatures. 

For example:

Set your machine to dwell at 37C (or whatever temp the enzyme prefers) and you are good to go. 

If you have a double digest that includes a thermophile enzyme, like BstBI at 65C or one that likes cooler temps, ApaI at 25C, for example, program the thermocycler to dwell at the lowest temp for a period of time (1 hr is a good rule of thumb) and then automatically shift to the higher temperature (for another hour or so). 

You can even set the machine to that shift to 4C and refrigerate the reaction until you are ready for it. 

All hands off, all done automatically.

If your restriction enzyme(s) is(are) inactivated by heat, you can set the reaction to “cut and kill” by incubating the reaction at the permissive temperature and then automatically raising the temp to destroy the enzyme(s). This can be useful since some enzymes can associate with DNA during the agarose gel electrophoresis and alter the band mobility.

You can also get a similar effect by adding SDS to your gel loading buffer.

But it isn’t just restriction digests that can be done, just about any enzyme reaction to modify DNA/RNA can be done in a thermal cycler.

For example: phosphatase treatment, phosphorylation with poly nucleotide kinase, ligation, reverse transcription, (to name a few) and more, are easy to set up in a thermal cycler.

Why limit it to nucleic acids? Protein work is also easily done using a thermal cycler. 

One example:  “boil” your SDS PAGE samples in the thermal cycler, instead of using a boiling water bath.  Assuming your cycler has a heated lid, using the thermal cycler will prevent evaporation and the samples won’t “pop” open. 

You can also use the cycler to cool the sample after denaturing so that you don’t risk burns.

Trying to determine the effect of temperature on your favorite enzyme's kinetics? The thermal cycler has a perfect footprint for a 96-well or 384 well plate to do the kinetics under controlled temperatures. 

Even if you aren't concerned about different temperatures, having your samples consistently at one temperature makes separate experiments more reproducible.  

Bonus:  If your thermal cycler has a "gradient" option (where the thermal cycler creates a gradual temperature gradient across the block), then you can test multiple temperatures within the same experiment.

The 96-well footprint also makes it easy to do higher throughput westerns and homogenized assays, since it is the correct alignment for multi-channel pipettors.  

We hope that the first Tips and Tricks was helpful, but that just scratched the surface of the power of PCR.   Keep reading for more advanced techniques that will allow you to exploit PCR for everything it is worth.  We welcome the opportunity to share these with you. 

It's Tips and Tricks Tuesday!

Everyone knows that PCR is a great way to amplify DNA, but with careful design of the oligos you can add mutations of all kinds.  For example, point mutations are best added in the middle of the oligo (if possible), but larger mutations, such as sequence to add an expression tag (like the 6x His tag) should be part of the 5’ end of the oligo.  In fact the 5’ end of the oligo can have a great deal of flexibility in its sequence, as long as the 3’ end has perfect matching sequence.

Remember though, when introducing mutations, the Tm of the oligo will change.  At first, the oligo will anneal only to the 3’ end with the perfect match, but, after a few rounds, there will be newly synthesized template that has incorporated the mutation oligo, so the annealing will now be the entire sequence of the oligo, not just the 3’ end.  You can optimize your PCR conditions by running a 2 phase run.  For a few rounds (5-10) assume that only the matching 3’ end of the oligo will anneal and calculate the Tm accordingly.  After that, switch the annealing temp (or switch to a 2 step PCR if the oligos allow it) to take into account the higher Tm of the entire oligo annealing.

Single point mutations are useful, but you can use the same concept for making libraries of different sequences. For example, a common tactic is to replace a particular amino acid in a protein with every other option (so 20 different possibilities -19 mutants and 1 wild type).  You don’t have to synthesize 20 different oligos to do that, thanks to the degeneracy of the genetic code (and some creativity) you can get all 20 codons with about 7 different oligos (even fewer if you eliminate certain amino acids that are likely to be really problematic like proline).

Hit us up for the list of 7 sequences that hit every amino acid codon 1 time each.

We are happy to help!

Just like you can add mutations or “tags” to the ends of the sequence, you can also add restriction sites to the ends of the sequence for directional cloning into your vector. Remember to add 6 bases between the end of the oligo and the new restriction site so you can digest the PCR product and ligate directly (versus cloning the product into a TA, or similar, vector first).  Even if you purify the product, digestion at the ends of PCR products can be inefficient so digest longer than you normally would to ensure sticky ends.  Otherwise it will act like any other vector/insert ligation (and save you several days of sub cloning and shuttling from one vector to another).

Larger mutations (dozens of bases) can be introduced into coding sequences without a local restriction site through PCR.  This will require a few steps and can get complicated.  Send us a message if you need clarification.

Basically, you will set up multiple PCRs.

1. From the 5' end (using  a convenient restriction site as a starting point-see tip 3) to the region to be mutated.
2. From the area to be mutated (where PCR 1 ended) to the 3' region of the final desired sequence (typically terminating with a different restriction site).  Note the antisense oligo from PCR1 and the sense oligo of PCR2 will be reverse compliments of each other.
3. Run the 2 PCRs and collect the fragments.  The 3' end of PCR1 will have the same sequence as the 5' end of PCR2.
4. Mix the two fragments and add the sense oligo from PCR1 and the antisense oligo from PCR2 in PCR3.  The two fragments will hybridize with their identical sequence and fuse together into 1 product.
5. The final product of PCR3 will be your final sequence with the mutations in the middle.

This method can be used to fuse any sequences together, so get creative.  You can also repeat the same technique to fuse more than 2 sequences together, just repeat the process for each pair and then link together.  Try to reduce the number of separate PCRs (to limit mutations) by doing pairs together and then fusing those products, rather than one at a time.

SO if you are going to put 4 pieces together, then fuse 1 and 2 together and fuse 3 and 4 together, then fuse 1-2 with 3-4.  It is also possible to put together more than 2 pieces at a time, but the optimization gets much more difficult.  Doable, but increasingly harder.

Finally, you can integrate any insert into any vector by PCR (without any restriction sites). It takes a bit of planning up front.

• First, decide if you are going to fuse the 5’ or 3’ end of the PCR product (assume the 5’ end will be fused for this example, but it doesn’t matter really).
• Second, determine the point where the insert will be fused into the vector (this can be basically any point).
• Third, design a sense oligo to the vector, starting at the insert point and going 3’ away from that point
• Fourth, design an antisense oligo to the vector starting at the insert point and going 5’ away from that point (I know that sounds confusing but imagine you are making a PCR product of the entire vector so that it is linearized at the insert point) This oligo has to have complimentary sequence to the 5’ section of the insert.
• Fifth, design a reverse compliment oligo (sense) to the one you made in the fourth step.
• Sixth, amplify the vector with the oligos from the third and fourth steps (this will be a long PCR depending on the size of your vector). Amplify the insert with the oligo from the fifth step and the 3’ oligo from however you made the insert.
• Seventh, mix the two PCR products and then fuse them together like you do in tip 4. This produces a linear piece of DNA that has the vector and insert attached at the insertion point.
• Finally, purify the product and add ligase. The intra (within one molecule) ligation is preferred over the inter (between two molecules) ligation so you will end up with nicked circles.  Even if you don’t phosphorylate the ends first, this works as the bacteria will repair the nicks once you transform them.  It sounds complicated, but it is really just an extreme version of tip 4.

The team here at Modern Vector have decades of experience with molecular biology.  Over all of that time we have picked up a bunch of rules of thumb and protocols to make doing the science easier, faster and cheaper.  We welcome the opportunity to share these with you in a new series, Tips and Tricks Tuesday!

The polymerase chain reaction (PCR) has been around for decades and is easily one of the most widely adopted technologies in biotech.  The power of PCR makes it extremely versatile and quite helpful.  However there are a bunch of things that you can do with PCR that may not be obvious.  Time for a bit of “insider info”.  Enjoy!

Own your next PCR

Tm is the temperature where 50% of the oligo is annealed to the template assuming complimentary sequence.  There are calculators to determine Tm of any sequence, but the rough approximation of 2 degrees for each A or T base and 3 degrees for each C or G base works well enough.  This only breaks down when you have mismatches and the “bubble” (or non-annealed double stranded sequence) affects the affinity of surrounding bases.  A few mismatches clustered in the 5’ half of the oligo have less impact on Tm than mismatches in the 3’ half of the oligo.  Design carefully.

Hot start polymerases are popular since they theoretically do not have any activity until exposure to high temperature removes the inhibitor (commonly an antibody or aptamer).  Without activity at lower temperatures, the enzyme is less likely to produce off target effects, but they tend to be more expensive than non-hot start enzymes. 

You can replicate this by adding a hold step at 95 - 98C (whatever the recommended denaturing temp for your enzyme) to your cycling parameters so the block is at max denaturing temperature.  Then keep your reaction very cold while setting it up (an aluminum block stored in the fridge works really well – also acts as a rack - don't put the block in the freezer, it will be too cold and could freeze your reaction).  By transferring the cold reaction to the preheated block, the temperature of the reaction will rise so quickly that the polymerase will not have a chance to do anything before the temp rises above the annealing temp of any off-target products. (And you will save some budget too.)

I know there is a great deal of opinion about there about primer design, optimal primer length, GC content, etc.  But, long primers can make PCR runs really fast.  Most companies have a set price for oligos up to certain lengths.  By maximizing the length (or getting close to it) you raise the Tm of that oligo.  If you get the Tm close to the extension temperature of the polymerase you are using (typically between 65 and 72 C – depending on the polymerase – or blend of polymerases – you are using) you can run the PCR as a 2 step instead of a 3 step (since you no longer need an “annealing step”). 

Cutting that extra step out of your cycling greatly reduces the time you need for the run.  2 step PCRs can also have higher specificity by reducing the chance that non-optimal interactions between oligos or oligos and template have a chance to form. (Unexpected amplifications are typically the result of weak polymerase activity at lower temperatures.  2 step PCR eliminates those lower temps.)

Unless you have no other choice, do not end your primers (the 3’ end of the oligo) with 3 Guanine and/or Cytosine in a row.  As the polymerase binds to the oligo/template hybrid, the 3’ end is critical for extension.  A mismatch in the last 3 bases will greatly reduce the chance of extension (you can use this to create SNP detection oligos where the 3’ most base will only match one variant).  But conversely, having 3 (or more) Gs and Cs at the 3’ end (due to their higher affinity) can stabilize the annealing of an oligo to an off-target template allowing for polymerase extension even if the anneal isn’t perfect.  It is best to generate the oligo with the 3’ end having 1 or 2 Gs and Cs in the last three to minimize the GC-clamp effect.

Everyone knows that PCR is a great way to amplify DNA, but with careful design of the oligos you can add mutations of all kinds.  For example, point mutations are best added in the middle of the oligo (if possible), but larger mutations, such as sequence to add an expression tag (like the 6x His tag) should be part of the 5’ end of the oligo.  In fact the 5’ end of the oligo can have a great deal of flexibility in its sequence, as long as the 3’ end has perfect matching sequence.

Remember though, when introducing mutations, the Tm of the oligo will change. At first, the oligo will anneal only to the 3’ end with the perfect match, but, after a few rounds, there will be newly synthesized template that has incorporated the mutation oligo, so the annealing will now be the entire sequence of the oligo, not just the 3’ end. 

You can optimize your PCR conditions by running a 2 phase run.  For a few rounds (5-10) assume that only the matching 3’ end of the oligo will anneal and calculate the Tm accordingly.  After that, switch the annealing temp (or switch to a 2 step PCR if the oligos allow it) to take into account the higher Tm of the entire oligo annealing.