Protein Engineering Protocols - Mycobacteriology research center

Compartmentalized Self-Replication 2453. Centrifuge the sample at 20,000 rcf (13 krpm) for 10 min at room temperature ina benchtop centrifuge. Discard the supernatant (containing unincorporatedprimers and dNTPs) and resuspend the pellet in TE (see Subheading 2., item 14).4. Unacceptable carryover of unselected polymerases can be further reduced byadding 20 to 50 U DpnI to the aqueous phase extracted using ether, and incubatingfor 1 h at 37°C (see Note 4).5. To ensure complete removal of primers, further purify CSR products from theaqueous phase using a PCR purification kit (Qiagen). Include two washes with35% (w/v) guanidinium hydrochloride during purification to ensure completeremoval of any residual primers. Elute purification products in 50 µL of providedbuffer EB (Qiagen; see Subheading 2., item 16).3.5.2. Quick Work-Up1. As in Subheading 3.5.1., step 5 purify CSR products from the aqueous phaseusing a PCR purification kit (Qiagen). Include one wash with 35% (w/v) guanidiniumhydrochloride during purification to ensure complete removal of any residualprimers. Elute purification products in 50 µL of provided buffer EB.2. Take 7 µL of the elution product and add 1 µL of 10X Taq buffer, 1 µL of 20 U/µLDpnI, and 1 µL ExoSAP, and incubate for 1 h at 37°C and 15 min at 85°C.3.6. Pull Through1. Rescue purified selection products by reamplifying using out-nested primers 3 and 4.Typically, 20% of purified selection products can be carried over into 50 µL of arescue PCR reaction (i.e., 10 µL out of the 50 µL of purified products). However,it may be advisable to use more or less depending on the initial result (see Notes).The cycling conditions for reamplification should be optimized according to theyield of the selection products, typically 25 cycles with the profile 94°C (30 s),50°C (30 s), and 72°C (5 min) will yield a strong reamplification band. The numberof cycles may need to be adjusted depending on the result (see Notes).2. Visualize rescued selectants using standard agarose gel electrophoresis. A successfulselection is indicated by the presence of a band of the correct size (2.5 kbp, inthe case of Taq polymerase) in the selection, and the absence of such (usually theabsence of any band) in the negative control (see Notes 5 and 6).The CSR band can be further gel purified, restricted, and recloned into the vectorpASK75 using standard procedures. After transformation into TG1 (seeHeading 2., item 1), transformants can be either screened as in Subheading 3.7.or pooled, grown, and induced for a further round of selection, as described inSubheadings 3.2.–3.6.3.7. Quick Screening of Taq Polymerase Variants1. To screen rescued selectants, transformed colonies are replica-spotted on TYE-Amp plates (see Subheading 2., item 3) and inoculated into a 96-well tissue cultureplate containing 100 µL of 2X TYA (see Subheading 2., item 2) per well.