Protein Engineering Protocols - Mycobacteriology research center

Synthesis of Libraries and Screening With the DHFR PCA 267(10–16 cycles). The product of the PCR is a linear version of the plasmid withoutthe deleted region. After the PCR, the resultant reaction mixture is digested withDpnI to digest the template DNA. After ethanol precipitation, approx 1/10 of thePCR product is ligated (25 ng PCR product/50 µL ligation reaction) and transformed.The positive clones are screened for the insertion of the appropriaterestriction site. A frame shift is included to reduce the possibility of stop codonread-through. The PCR protocol is derived from the ExSite PCR-based sitedirectedmutagenesis kit (Stratagene).2. We used Taq polymerase because the sequences we wanted to amplify were relativelyshort. For longer genes, we suggest Pfu or Pfu Turbo polymerase, whichgive more reliable results than Vent polymerase in our hands with this protocol.If the difference in size between the product of PCR 1 and PCR 2 is too large,thus particularly for large genes, we would recommend a mega-primer protocol(50,51). Both approaches with slight modification could be useful to generatecombinatorial libraries in which regions far apart in the sequence are variedsimultaneously.3. The Gelstar is diluted 1 × 10 –4 to 5 × 10 –5 in TAE agarose gel. Gelstar is muchmore labile than ethidium bromide and, thus, the gels should be prepared on theday that they will be used. In addition, it is difficult to easily quantify DNA withGelstar because the signal becomes saturated at lower quantities of DNA, and,thus, it is difficult to distinguish different amounts of DNA above a certain threshold.In addition, one must be careful not to load large amounts of DNA per wellbecause this could dramatically affect the migration of the samples (typically, wedo not load more than 300 ng of plasmid DNA per 25-µL well). Gel staining withethidium bromide also permits visualization of DNA with the blue light of theDark Reader, although more DNA must be loaded per well (>1.5 µg for digestedplasmid DNA) to allow for visualization of small fragments or PCR products. Inthe worst case, ultraviolet ethidium bromide visualization can, of course, be used,but care should be taken to process the bands as quickly as possible.4. The QIAquick gel purification protocol is preferable for generating severallibraries at the same time, but is more expensive. As an alternative to the proceduredescribed in Subheading 3.2., step 3, the electrophoresis step can bereplaced by a 2-h, 37°C incubation of the PCR product with the restriction enzymeDpnI to remove the plasmid template DNA. Then the PCR products are purifiedaccording to the QIAquick PCR purification protocol.5. This amount could be increased in proportion to the size of the gene under study.The relative quantities of product from PCR 1 and PCR 2 added to PCR 3 areadjusted according to their relative molecular weights.6. The number of amplification steps in PCR 3 has to be minimized because failureto do so could be detrimental to library representation. For this reason, the numberof cycles is minimized and the concentration of the two terminal primers isreduced (see Fig. 3). It is worth optimizing this step to obtain the maximum quantityof product in a minimum number of cycles. We recommend using Taq polymerase,as described in Note 2.