X. phage

Figure 4.5. Integrative and excisive X phage recombination pathways. Integration is catalyzed by the X Int protein in a reaction that also requires the E. coli IHF protein. Recombination occurs within a common core sequence of 15 base pairs. The excision reaction requires the X Xis protein in addition to Int and IHF.

Bustamante C, Marko JF, Siggia ED et al (1994) Entropic elasticity of X-phage DNA. Science 265 1599-1600... 

Figure 28-11 Genetic and physical map of the X phage genome. After Szybalski. See Honigman et al.255 for a more detailed diagram of the immunity region. The gene for the lambda repressor is labeled C[.

Standard curve obtained by electrophoresis of X phage DNA fragments from EcoRI cleavage. 

Standard restriction enzyme digest, X. phage DNA digested by roRI Constant-temperature water bath at 37°C... 

Load one sample into each of the sample wells in the following manner. Mix each 20 fiL of reaction mixture from the restriction enzyme with 10 fxL of gel-loading buffer and apply one 10 nL sample to each well. Put 10 fiL of roRI standard digest of X phage DNA into one sample well. 

Describe the pattern obtained from electrophoresis of a standard coRI digest of X phage DNA on 2% agarose gel. 

Disposable gloves should be worn when you are handling the enzyme container. Remove the enzyme from the freezer just before you need it. Store the enzyme m an ice bucket when it is outside the freezer. The enzyme should never be stored at room temperature. Because of high cost, digestion by restriction enzymes is carried out on a microscale level. A typical reaction mixture will contain about 1 fig or less of DNA and 1 unit of enzyme in the appropriate incubation buffer One unit is the amount of enzyme that will degrade 1 fig of X phage DNA in 1 hour at the optimal temperature and pH. The total reaction volume is usually between 20 and 50 fiL. Incubation is most often carried out at the recommended temperature for about 1 hour. The reaction is stopped by adding EDTA solution, which complexes divalent metal ions essential for nuclease activity. 

A non-contact heating method called infrared-mediated temperature control has been employed for PCR. Because the chip material (polyimide) does not absorb IR, only the solution absorbs IR. Therefore, the low thermal mass of the solution allows for fast thermal cycling, and 15 cycles have been achieved in 240 s Amplification of X phage DNA (500 bp) was first conducted at 94°C for 10 s, followed by 15 cycles of 94°C (2 s), 68°C (2 s), 72°C (2 s), then finally stopped at 72°C for 10 s [192],... 

In another report, PCR and subsequent CGE separation were integrated on a glass microchip (see Figure 9.1). PCR of X phage DNA was conducted in a sample reservoir with thermal cycling by a Peltier heater/cooler. Subsequent CGE separation was conducted immediately after PCR because the PCR reservoir led to the CE channel. In addition, an on-chip DNA pre-concentration device was included. This reduced the analysis time to 20 min (by decreasing the number of thermal cycles required to 10 cycles), and the starting DNA copy number to 15 (0.3 pM) [925],... 

In another report, a PDMS rotary device (12 nL) was used for PCR both spatially (moving over three temperature zones) or temporally (at three temperatures with rotary mixing). PCR of a P-actin gene (123 bp) and X phage DNA (199 bp) were demonstrated. Real-time PCR was also performed as detected by an intercalating dye (Sybr Green I) [357]. 

According to the opponents, the main argument against granting a patent was the lack of novelty of the recombinant DNA sequences claimed, which included the DNA sequences that coded interferon-alpha (IFN-alpha) as an insert. This prior art referred to DNA genome libraries, which are fractioned segments (15-20 Kb) of chromosomal DNA of human fetuses, cloned as derivatives of recombinant X phages. 

Maruyama IN, Maruyama HI, Brenner S, kfoo a X phage vector for the expression of foreign proteins, Proc. Natl. Acad. Sci. USA, 91 8273-8277, 1994. 

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