Cloning Brachyury from SW480 in pNEB193 Plasmid - Lab Report Example

Cloning Brachyury from SW480 in pNEB193 plasmid 8 Preparation of LB Ampicillin Agar Plates The LB Ampicillin agar plates weremade and kept for use in the blue/white screening. 8 – 2. Purification of RNA from Human Cells SW148 For this experiment, RNA was extracted from SW480 human cells that contain the Brachyury gene. This was the first step towards cloning the Brachyury gene. The Qiagen RNeasy Plus Micro Kit was utilised to determine the RNA. The protocol that was used is ideal to obtain RNAs more than 200 nucleotides long as shown in table 1. The sample (F) had an average concentration compared with other samples in the class. It had more than 0.470ng RNA; this permitted the continuation of the experiment to the next step. The standard deviation of the whole class data was 1.4. This was a small value which indicated that the concentration values obtained by the class had a small variation among them. This may have been because the conditions for the experiment were the same with small variations among them. As for my part, accurate pipetting meant that my results were more accurate than the rest. Also, the samples H, M also had higher concentrations than those of other students. For the quality of the RNA that was determined in the experiment to be analysed, denaturing agarose gel electrophoresis was utilised to view the bands 18s and 28s. The two kinds of RNA are utilised to evaluate the quality of mRNA that was selected in the experiment. 8 – 3: Synthesizing cDNA from the determined RNA This step which involved making DNA from the RNA that was determined in the first step was very important. The RNA templates which were used at the start of the experiment were taken from the samples. Using a SuperScript III system and the primers Oligo (dT)20, each student synthesised a cDNA from 741ng of RNA. The concentration of the synthesised cDNA was measured using a Nanodrop. The results are shown in table 2. The Concentration of cDNA produced from the RNA samples Student number Date and Time Nucleic Acid Conc. Unit A260 A280 260/280 260/230 Sample Type Factor A 07/03/2014 15:12:41 728.1 ng/µl 22.064 13.549 1.63 1.7 ssDNA 33 B 07/03/2014 15:13:26 747.1 ng/µl 22.64 13.845 1.64 1.74 ssDNA 33 C 07/03/2014 15:14:03 851.6 ng/µl 25.805 15.519 1.66 1.86 ssDNA 33 D 07/03/2014 15:14:37 727.4 ng/µl 22.043 13.527 1.63 1.75 ssDNA 33 E 07/03/2014 15:15:11 858.0 ng/µl 25.999 15.639 1.66 1.87 ssDNA 33 F 07/03/2014 15:15:38 302.3 ng/µl 9.161 6.574 1.39 0.88 ssDNA 33 G 07/03/2014 15:16:08 760.9 ng/µl 23.057 14.219 1.62 1.74 ssDNA 33 H 07/03/2014 15:16:40 724.6 ng/µl 21.958 13.564 1.62 1.72 ssDNA 33 I 07/03/2014 15:17:11 616.8 ng/µl 18.69 11.805 1.58 1.72 ssDNA 33 J 07/03/2014 15:17:36 760.8 ng/µl 23.055 14.095 1.64 1.77 ssDNA 33 K 07/03/2014 15:18:06 751.9 ng/µl 22.785 13.911 1.64 1.75 ssDNA 33 L 07/03/2014 15:18:50 639.4 ng/µl 19.375 11.835 1.64 1.77 ssDNA 33 M 07/03/2014 15:19:20 781.0 ng/µl 23.667 14.452 1.64 1.76 ssDNA 33 N 07/03/2014 15:19:52 801.4 ng/µl 24.285 14.919 1.63 1.73 ssDNA 33 O 07/03/2014 15:20:19 796.3 ng/µl 24.131 14.941 1.62 1.75 ssDNA 33 P 07/03/2014 15:20:50 816.9 ng/µl 24.754 15.208 1.63 1.77 ssDNA 33 Q 07/03/2014 15:21:27 788.6 ng/µl 23.896 14.689 1.63 1.76 ssDNA 33 R 07/03/2014 15:22:20 785.5 ng/µl 23.803 14.436 1.65 1.79 ssDNA 33 S 07/03/2014 15:23:33 957.3 ng/µl 29.009 17.697 1.64 1.81 ssDNA 33 average 747.2 median 760.9 STDEV 131.2 Table 2: It shows the data obtained by the class for the measured concentration of cDNA using a Nanodrop. The average, median and standard deviation are shown. The concentration of my sample F was 302.3ng/μL. This was a very low concentration in comparison with the average of the class. Additionally, the standard deviation was 131.2. This meant that all the data obtained tended to close to the mean. Hence, the class did quite well in the experiment. 8 – 4. PCR Amplification of Brachyury from cDNA Special primers were used to amplify the total length of Brachyury obtained form cDNA which was formed in the previous step. The length which was expected for Brachyury was 1146bp. 8 – 4. PCR Amplification of Human β – actin The explanation for this step was to test whether the cDNA was appropriate for the PCR. Due to this, two primers were used to amplify the β – actin. This produced 353bp of the PCR product. Hence, the end product of the PCR was made up of the total cDNA length and there was no more amplification for the genomic DNA in the sample. 8 – 5. Agarose Gel Analysis of the PCR Products On evaluation of the agarose gel that had 0.8 % concentration by use of image lab, it was discovered that lane 1 had 3 faint bands that were 1325 and 1400bp at the top by approximation. This seemed to be a good result as Brachyury was amplified by primers from cDNA. Nonetheless, a second band was observed. This indicated that there was more than one template that could be connected to another kind of Brachyury which was in the established cDNA from the previous step. Band number 3 which was quite faint was the primer – dimer. A very bright single band (333bp) could be observed in lane 2 where β – actin was loaded onto the gel. This signified that cDNA was appropriate for PCR amplification. 8 - 6. Purification of DNA from the PCR Reaction Mixture Sigma GenElute cleanup kit NA1020 was used to purify the DNA fragments obtained from the PCR product. It is a suitable kit that can be used to purify DNA fragments as in this experiment, nucleotides, excess primers and other compounds that are between 100 bp to 10 kb. On purification, the products that are obtained may be used for cloning or microarray. 8 - 7. Restriction Digestion for PCR Purified Fragments and pNEB193 Plasmid Plasmid pNEB193 was availed. It was to be used to insert the Brachyury gene. EcoR1 enzyme was utilised to digest the PCR product and the plasmid pNEB193 so as to finish the experiment. To hinder self – ligation, alkaline phosphatase was utilised to treat the digested plasmid. Self – ligation was hindered by taking away the phosphatase group from the 5’ prime end at the sliced end of the plasmid. 8 – 8. Purifying the DNA from the Agarose Gel In this step, gel purification was done to purify the linear phosphatase treated plasmid DNA molecules from any circler plasmid molecules. The linear molecules were for use in the ligation reaction. They were also to be used in purification of the PCR fragments. To make sure that the appropriate length of the PCR fragment was cloned, the linear molecules were also used. Nevertheless, cloning was tried for everything that was amplified in the Brachyury PCR reaction. This is shown in figure 3. It can be seen from figure 3, lane 2 that there were 2 bands. The first band was nicked, approximately 3650bp. The second one was around 1794bp. This meant that the control plasmid reaction was correct and that the plasmid was uncut. In the third lane where the EcoR1 digestion/phosphatise treat were loaded, a single thick shiny band that was used in the ligation reaction step was spotted. This band had a size of 2750bp. In the last lane where the purified PCR product was loaded, EcoR1 enzyme functioned effectively and divided it into 2 bands. The first band was the product of the PCR, while the second band was the primer – dimer. The shiny band was excised using the x – tracta Gel Extraction Tool which was utilised in the ligation experiment. 8 – 9. Estimation of Molar Ratio via Gel Analysis of DNA Fragments The molar ratio is an extremely vital parameter which tells the efficiency of the digestion of Brachyury and the plasmid. It is used to determine this efficiency. It also is vital in the planning of the ligation reaction. The molar ratio was calculated by the demonstrator using the Image Lab version 4 as shown in figure 4. In this step of the experiment, 1% agarose gel was used by the demonstrator to evaluate the DNA fragments. Hyper ladder was loaded in lane 1, digested plasmid that was 5μl in lane 2, and 5μ l of the digested PCR product in lane 3. It was clear that several students were confused as they handed in plasmid tube instead of the PCR tube to the demonstrator. This explained why there were two faint bands besides the two shiny bands in the second and third lane. 8 – 10. Ligation of EcoR1 Digested Purified Brachyury PCR Fragments and EcoR1 Digested Phosphatase treated Gel Purified pNEB193 Plasmid In this step, ligation of the PCR fragments to the pNEB193 digested plasmid was tried so as to form a new recombinant plasmid. The new transformed plasmid was used in the transformation step. 8 – 11. Transformation of the Chemical Competent E. coli cells with Plasmid DNA A minute aliquot of the ligation reaction was added to the ‘chemically competent’ E. coli cells. The motive for this step was to insert the whole ligated plasmid which was formed. The E.coli cells that were probably transformed were culture on LB ampicilin agar plates that were prepared in step 1. 8 – 12. Preparation of LB X – GEL/ IPGT Plates for White/Blue Screening Two control plate were cultured and labelled control 5 and control 100 along with four ligation plates; Ligation 20, 40, 80 and 160 as shown in table 3. The four plates were incubated throughout the night and checked for the white and blue colonies the following day, the results of which are shown in table 4 and table 5. The white/blue screening technique based on the bacteria that had the full insert would not calve X – gal/ IPGT in the media. Hence, the growth of the white colonies on the plates meant that the transformation had been fully formed. In contrast, the growth of blue colonies is regarded as negative transformation (Langley, K.E et al. 1975). Student number blues on L20 plate Brachyury insert clones/ng DNA A 220 21 B 570 22 C 387 3 D 437 2 E 0 0 F 230 15 G 848 11 H 200 11 I 0 0 J 966 60 K 408 16 L 752 79 M 1652 53 N 224 42 O 1816 41 P 300 53 Q - - R 942 26 S 800 17 Totals 10752 472 Average 597 23 standard deviation Table 3: Data for the transformation in the ligation reaction step for all students. Table 5 shows the data for the ligation reaction established by the class. It shows the total count of the colonies, average and the standard deviation. The efficiency of transformation can be calculated from the table. In regard to control 5, the efficiency of transformation is 1.38696e2transformants/ug DNA or 1.38696 e – 1 transformants/ng DNA lest there are no more dilutions added to the transformation reaction after the DNA. For the ligation reaction 60, it was 8.38889e – 1 transformants/ug DNA. 8 – 13. Colony PCR to check for Brachyury Positive Recombinant Plasmid in E.coli Colonies In this part of the experiment, White colonies that were cultured by the demonstrator were used. Colony PCR was utilised to evaluate the white colonies for a recombinant plasmid. The reason why colony PCR was used is because it is quite fast, sensitive and best for screening of numerous colonies of bacteria that might have been tentatively the anticipated length of the Brchyury gene in its modified plasmid. The products of the PCR were then loaded onto 0.8% agarose gel for about one hour. The bands produced were then evaluated using ImageLab software as shown in figure 5. After analysing the products of the colony PCR as shown in figure 5, it was evident that a very bright band was present on each lane. ImageLab software was the utilised to compute the tentative size of each band. Lane 2 had a band of 1380 bp, lane 3 had a band of 1420 bp, lane 4 had a band of 1440 bp and lane 5 had a band of 1445 bp. The sizes of the bands as depicted by the ImageLab software showed that the white colonies may have had the correct size of the insert. Thus, the huge majority of the vectors may have contained a full length of the Brachrury gene. Further experimenting such as plasmid mapping and sequencing is required to continue this experiment and ascertain the outcome. Bibliography Langley, K.E. et al., 1975, ‘Molecular basis of beta-galactosidase alpha-complementation,’ Proceedings of the National Academy of Sciences of the United States of America, 72 (4), pp.1254–1257. Read More