Results and Discussion
An unknown gene was amplified, cloned, sequenced and identified as Hog1 out of a vector. Hog1 was amplified by PCR and analyzed on a 1.5% agarose gel (Figure 1). Lane 2 shows the Hog1 band at ~2 Kb; Hog1 has an expected size of 1.8 Kb but this discrepancy is within sample error/variation of AGE. Lane 3 shows a very faint band at ~2 Kb that is likely overflow from Lane 2. The absence of DNA in lane 3 indicates our sample was not contaminated and supporting successful Hog1 amplification. DHα was transformed with the pRS306 vector, amplified in LB/AMP media, extracted by miniprep then analyzed on AGE (Figure 1, Lane 4). Lane 4 shows a bright band at 5 Kb – within sample error/variation of the 4.5 Kb expected fragment size – indicating amplification was successful. Figure 1 results were supported by nanodrop absorption (data not shown) in preparation for Hog1 and pRS306 ligation.
After pRS306 and Hog1 amplification, each sample was digested with BAMHI-HF and XbaI to create complimentary overhangs for ligation. The digests were separated and analyzed via AGE (Figure 2). pRS306 was digested with BamI and XbaI separately (lane 3 and lane 4). Each single cut digest migrated in between the nicked and supercoiled DNA of the uncut vector (lane 5) indicating BamI and XbaI successfully cut the plasmid. The nicked and supercoiled band in the XbaI digest (lane 3) indicates that XbaI was not 100% efficient in cutting the vector. Nonetheless, most of the vector was cut enabling and was not a concern. Lane 6 shows the Hog1 band at ~2.5 Kb. Due to the small size of the cleaved nucleotides on the insert, it is not possible to confidently assess insert digest efficiency with an internal control. However, both enzymes cut the vector (Lanes 3 and 4) and a band appeared near the expected size (Lane 3) providing evidence the insert was cut. This evidence of the successful digest prepared the fragments for ligation.
After the pRS306 and HogI digests were ligated, DH5α competent cells were transformed with the ligation products. The colonies grew as expected (Table 2) with the no vector (H2O) control yielding 0 colonies demonstrating the sample was not contaminated and the positive pUC19 vector produced a lawn indicating the transformation protocol was successful. The no insert column indicates the background ligation rate – that is the vector that closed on it’s self without the insert. A low background 2 colonies indicates the 1:3 vector:insert plate has one false positive for every 150 positive ligations. The 1:7 ligation ratio also grew successfully with 85 colonies. The high ligation rate and successful transformation indicated the products were ready to be amplified and sequenced.
Four colonies were amplified in LB/AMP cultures. pRS305-Hog1 was isolated and digested with XbaI and BamHI to screen for the Hog1 gene and pRS306. An AGE of the digest shows the presence of a faint Hog1 band in each colony (Figure 3). Each colony shows a...