RECEPTOR
2.2 Materials and Methods
2.2.3 Cloning of gel purified ECD amplicons into pTZ57R/T Vector
Figure. 2.3: Detailed vector map of pTZ57R/T vector.
2.2.3.2 Ligation of gel purified EGFR ECD amplicons into pTZ57R/T vector
The gel eluted PCR amplicons of EGFR ECD were ligated to pTZ57R/T vector following the components of ligation reaction as mentioned in Table 2.2. The ligation reactions were setup in 0.5 ml microcentrifuge tubes and incubated at 4°C overnight to get maximum number of transformants. The reactions were setup at an insert: vector molar ratio of 3:1, where the amount of insert required in a reaction is calculated using the following formula
Amount of vector (ng) × Size of insert(kb)
× Insert :Vector molar ratio = Amount of insert (ng) Size of vector (kb)
Table 2.2: Components for ligation reaction setup for TA cloning of PCR amplified ECD amplicons.
Reaction components Volume (µl) 10X Ligation Buffer 1.0
pTZ57R/T Vector (50ng) 1.0 (55ng)
PCR product 2.7 (110 ng)
T4 DNA Ligase (3 units/μl) 1 Nuclease-free water 4.3 Total reaction volume 10.0
2.2.3.3 Transformation of ligated products after TA cloning
After 16 hours of incubation, ligated construct was transformed into competent E. coli (XL10 Gold) cells.
2.2.3.3.1 Preparation of E. coli (XL10 Gold) competent cells Day 1
1. 50 µl of culture of E. coli (DH5α) from glycerol stocks were inoculated into 5.0 ml LB medium and incubated overnight at 37ºC and 180 rpm.
2. 0.1 M CaCl2 solution was filter-sterilized by passing through 0.22 µm filter in laminar air flow and kept in refrigerator.
Day 2
3. 1.0 ml culture from day 1 was inoculated into 100 ml LB medium and incubated at 37ºC with 180 rpm till cell OD reached 0.4-0.6 at 550 nm.
55 (ng) × 1.935 (kb)
× 3
= 110.628 ng
2.886 (kb) 1
4. The cultures were transferred aseptically to round bottom centrifuge tubes and centrifuged at 4ºC with 4000g for 10 min.
5. The cell pellet was first suspended in 3-4 ml sterile, ice-chilled 0.1 M CaCl2
solution followed by making up the final volume to 20 ml. The suspension in centrifuge tubes was kept on ice for 10 min.
6. The tubes were centrifuged again at 4000g at 4ºC for 10 min. The supernatant was carefully removed and the pellet was resuspended in 3.0 ml of sterile ice chilled 0.1 M CaCl2 solution.
7. 200 µl of competent cells were aliquoted into each 1.5 ml microcentrifuge tube containing 10- 15 (%, v/v) glycerol (final concentration) and kept at -80ºC for further use.
2.2.3.3.2 Transformation of ligated products after TA cloning
The E. coli (XL10 Gold) competent cells were transformed with the ligated product. The aliquots of competent cells (200 µl) was taken out from -80°C and kept on ice for 5 min, followed by addition of 10 µl of ligation mixture. The tube was gently tapped 4-5 times and kept on ice for 30 min. Then the cells were given a heat shock at 42°C for 90 secs.
The cells were immediately transferred to ice for 2-3 min. 800 µl of super optimal medium with catabolite repression (SOC) was added to the transformed cells. The transformed cells were incubated at 37°C in a shaking incubator at 180 rpm for 1h (Hanahan, 1983). The cells were harvested by centrifugation at 2000g at 25°C for 5 min.
800 µl supernatant was discarded and the cell pellet was resuspended in remaining 200 µl supernatant. The 200 µl transformed cells were spread plated on LB agar plates supplemented with antibiotics ampicillin, IPTG (Isopropyl β-D-1- thiogalactopyranoside) and X-Gal (5-bromo-4-chloro-3-indolyl-β-D-galactoside) at a final concentration of 50 µg/ml, 0.2 mM and 80 µg/ml, respectively. The LB agar plates were incubated overnight at 37°C.
2.2.3.4 Screening of positive TA clones of EGFR ECD
Positive TA clones were identified by blue white screening. Overnight grown plates were observed for blue white colonies. White colonies containing the positive clones were
picked up in a laminar air flow and grown overnight in 5 ml LB media supplemented with ampicillin (50 µg/ml). The positive TA clones produce white colonies are unable to produce β-galactosidase because of the interrupted lacZ gene induced by the presence of the insert DNA. In absence of β-galactosidase the cells cannot degrade X-Gal and produce blue colour colonies.
2.2.3.5 Isolation of plasmid DNA from positive colonies of TA clones by NID method Plasmid DNA was isolated from fully grown recombinant E.coli (XL10 Gold) cells which were picked up from the LB plates after transformation of the ligated TA cloned products of EGFR ECD. NID method as described below was employed for isolation of plasmid (Lezin et al., 2011).
Table 2.3: Composition of NID extraction buffer for plasmid isolation.
Components Final Concentration
Tris pH 8.0 50mM
Sucrose 5%
EDTA 50 mM
NH4Cl 75mM
Triton X-100 0.5(%v/v)
Lysozyme 100 µg/ml
RNase A 25 µg/ml
CaCl2 50mM
1. 1.5-2 ml of bacterial cultures were pelleted at 6000-7000 rpm for 1 min.
2. After drawing 150 µl extraction buffer into a pipette tip, the pellet was loosened off the tube wall with the tip without releasing the buffer. Then the extraction buffer was added and the pellet was resuspended.
3. The bacterial suspension was incubated at 65°C for 5 min.
4. Suspensions were centrifuged at maximum rpm for 10 min or until a tight bacterial pellet was formed. The pellet was removed with a toothpick.
5. 100-120 µl isopropanol was added, followed by mixing and centrifugation of the solution at 7000 rpm for 10 min at RT.
6. DNA usually forms film-like precipitates that adhere well to tube walls and are invisible in isopropanol solutions. After discarding the supernatant, the DNA was centrifuged after adding 70% ethanol. Ethanol was removed, and the DNA pellet was dissolved in 20-50 µl TE buffer.
A 100x enzyme stock containing 10 mg/ml lysozyme and 2.5 mg/ml of RNase A prepared in 50% glycerol and 50 mM Tris pH 8 was stored at −20°C and used repeatedly.
2.2.3.6 Screening of recombinant plasmid DNAs for identification of positive TA clones by colony PCR and Restriction digestion
1 µl plasmid DNA isolated from individual colonies were subjected to PCR amplification using ECD_F1 and ECD_R1 primers as described in section 2.2.2. The PCR amplicons were run on 0.8% agarose gel.
5µl of the recombinant plasmid DNA of PCR positive clones EGFR ECD were taken in a fresh sterile microcentrifuge tube for restriction enzyme digestion analysis. The recombinant plasmid DNA was digested with restriction enzymes, NdeI and XhoI, to check for positive clones following a 20 µl reaction mixture set up as mentioned in Table 2.4. The reaction mixtures were incubated at 37ºC in a water bath for 90 min. The digested products were then run on 0.8% agarose gel as described in Section 2.2.2.1. The digested vector and the respective insert DNA of above mentioned recombinant derivatives were visualized by placing the gel under UV transilluminator. The digested fragments of expected size were taken as positive TA clones. Glycerol stocks of E. coli (XL 10 Gold) cells containing the positive TA clones were prepared in glycerol (15-20
% v/v) and stored at -80°C.
Table 2.4: Restriction enzyme digestion set up of recombinant plasmid DNA of EGFR ECD.