Cloning Brachyury from SW480 in pNEB193 plasmid - Essay Example

Cloning Brachyury from SW480 in pNEB193 plasmid The SW480 cells were prepared and harvested in a cell culture flask. Hence, all the cells were removed and stored them in preparation for the RNA extraction. Examinations and recordings were done to examine if the cell count and cell morphology were allowed before the step of RNA extraction.The SW480 cells were observed under the microscope as small round cell. On checking the cells count and viability using TC BioRad counter, the following results were obtained:Slot A – Cell number – 2.

54 x 105cells/mLSlot B - Cell number - 2.30 x 105 cells/mL, viable cells: 2.30 x 105 cells/mL (100%)This number was partitioned into 2 tubes; each tube contained 1.84 x 106 SW480 cells. This number of cells was adequate to go on with the RNA extraction process. However, it is good to note that the recommended starting point is 3 x 106 cells per extraction.2-figure 2: The total RNA was extracted from SW480 cells by use of Norgen’s Total RNA Purification Kit, the samples were then denatured in rapid Formalin – free RNA loading buffer which had Formalin – Free RNA dye.

They were then incubated for 5 minutes at a temperature of 700C. Lane number one was filled with RNA ladder 5 μL. The lanes from number 2 to 20 contained 10 μL of each of the class samples. The image capture was then done using GelDocEZ system.3-Table 1: The concentration and purity of the total extracted RNA from the SW480 cells for the sample H was is shown in table 1. The resulting concentration of RNA was 82.88 ng/ul. An RQI of 7.3 indicated that the RNA quality was accepted. The ratio (28S/18S) was 0.

93 though the recommended ratio is 24 – Plasmid PreparationsThe plasmid preparation experiment was undertaken before the start of the RNA extraction. The purpose of this experiment was to purify enough linearized phosphatise treated pNEB183 plasmid to be utilised in the ligation reaction. This purification was attained through several steps that started with the purification of the inoculated plasmid from E.coli culture in LB/ampilicin broth. Using Qubit analysis, the concentration of the purified plasmid was calculated to be 5.1 ul. The EcoR1 enzyme was then utilised to digest the circular plasmid into a linear plasmid which was then treated using alkaline phosphatise enzyme to remove the 5’ phosphate group and hinder self – ligation.

The sample was then loaded on 0.8% agarose gel so as to visualize and purify the linearized plasmid from the gel by use of purification method as shown in figure 5:Figure: Using the GelDocEZ system imager, the purified phosphatise treated plasmid on 0.8% agarose gel was shown. The first lane is Easy Ladder 2, the Lane 2 is the circular and linear DNA plasmid. In figure 5, two bands were clearly seen in the second lane. The first band was an uncut plasmid. The second band was a linear plasmid.

Adequate preparation took place as the linear plasmid could migrate longer on the gel. The second band was bright and shiny. It was 2797 bps in length. It also contained .ul/mg concentration of plasmid. Using x tracta Gel Extraction tool and therefore Sigma GenElute Extraction kit, this band was excised for gel purification.However, it was noted that the final concentration of the linear phosphatise treated pNEB193 plasmid which was purified from the agarose gel was low in most of the student samples.

Various probable mistakes may have led to the white precipitation in the student samples. This was apart from the sample of student R which had a concentration of 22. The concentration of the plasmid of the sample of student R was verified for use in the ligation experiment.