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Day 6 Morning Lecture Notes

Steve Williams, Smith College

June 11, 2004

Subcloning from Lambda Phage to a Plasmid

Why transfer a foreign gene from lambda vector to a plasmid?

  1. Lambda DNA is linear and is therefore less stable than circular plasmid DNA. Once a gene has been cloned, we can store it indefinitely in a plasmid. The reason that linear DNA is less stable is the existence of exonucleases that attack the ends of DNA. Plasmids are not susceptible to exonuclease degradation.
  2. The insert makes up a greater fraction of the plasmid's DNA than it does of lambda's. Plasmids are typically about 3 kbp in size so RevT at 1.4 kbp will make up about 1/3 of the total. In contrast the lambda vector has about 39 kbp of DNA so the yield is 10x lower. Plasmid DNA is also easier to isolate than lambda.
  3. Plasmids are excellent templates for sequencing an insert while lambda are poor for this purpose. The reason why this is so is obscure.
  4. Lambda Zap Express can take an insert up to 12 kbp in length while plasmids can accommodate larger inserts.

Traditional, One-Week Method of Subcloning

  1. Pick positive lambda clone from a plaque and elute the phage into a buffer solution.
  2. Mix the phage with E. coli at the right ratio to get a good yield of phage.
  3. Isolate lambda phage via gradient centrifugation.
  4. Purify DNA from lambda phage via phenol-chisam extraction and then remove solvents via drop dialysis or ethanol precipitation.
  5. Cleave the insert out of the lambda phage via a restriction digest (here EcoR1).
  6. Run a check and check the digest composition. With EcoR1 and Lambda Zap Express, the gel should show bands for the left arm of lambda, the right arm of lambda and the insert. This step confirms that the previous reactions worked at the same time that it separates out the lambda DNA.
  7. Cut the insert out of the gel and purify it.
  8. Ligate the insert into a plasmid.

Note that steps 2 through 7 are necessary due to the low yield of phage from step 1.

New Subcloning Method using PCR

  1. Pick lambda clone from a plaque and elute the phage into a buffer solution.
  2. PCR amplify the insert.
  3. Ligate the insert into a plasmid.

There's an optional purification process between the first and second steps but only for sequencing, not for subcloning.

Special Vectors for PCR Subcloning

Vectors that are specially designed for convenient PCR subcloning are available. They produce a PCR product that has about 100 bp of lambda DNA at either end of the insert.

For some reason the products of Taq polymerase are always terminated with a single A at the 3' end. These PCR products can be terminated with vectors that have a single T overhang. EcoR1 overhangs will ligate only to other EcoR1 overhangs, while blunt ends are not at all specific. Single-A overhangs turn out to be fairly specific because naturally occurring single-T overhangs are unknown.

How to prepare single-T overhangs on plasmid DNA? Use a blunt-cutter enzyme on the plasmid and then employ DNA polymerase to add a T at each 3' end. A specially modified T NTP allows addition of only one base pair. After heat-killing the DNA polymerase, unblock the single T. In actual fact you can purchase a cut plasmid already terminated with the single T. The insertion into the plasmid is chose to occur at a place that doesn't interfere with an E. coli gene.

Note that the A overhangs don't ligate with each other. The only product should be the desired one except that not all the plasmid blunt ends will acquire T's, so there may be some plasmid without inserts as a background.

Quantitative Reverse-Transcription PCR

Lori Saunders, Gwathmey, Inc.

Formation of loops and unintended products like primer-dimers lowers the efficiency of PCR and causes problems. If the efficiency is 100%, the amount of product P = T*2n where T is the amount of starting RNA template and n is the number of cycles. More realistically P = T*(1 + E)n. Even so the efficiency will vary from cycle to cycle.

The goal of quantitative PCR is to back out the amount of template T (that is, the amount of gene expression) from P(n) data. Real-time or kinetic PCR is a technique that has been developed in the last 10 years to address this goal. The leading instruments are the TaqMan 7700 and 7000 developed by Applied Biosystems ($30-40K). In TaqMan the amount of product is detected very 7 seconds via fluorescence. The plot below shows the kind of raw data produced by real-time PCR. The product P is measured in fluorescence units. The complete real-time PCR experiment typically takes about 2 hours. The vendors who make PCR primers also sell fluorescent probes for real-time PCR.

As the initial amount of template T decreases, the cycle at which saturation occurs moves out to higher n. Due to noise, data at low n are unreliable. At high n all the curves for various values of T are the same. The steepest part of the curve is the best place to determine kinetic coefficients. Customarily one plots the amount of product at a given cycle versus T or the cycle at which P reaches a given value versus T (a "fixed fluorescence plot"). These plots can incorporate data for T values so low that saturation is never reached.

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