DNA TOPOISOMERASE I
Structure; The determination of crystal structure of a 67kDa and terminal fragment of E.coli topoisomerase I represents how these enzymes function.
Str-1
The 590 aa N-terminal fragment observed in the structure corresponds to the cleavage/strand passage domain. E.coli topo I contains four domains of protein.
Str-2
MECHANISM OF DNA RELAXATION
This mechanism has been referred to as enzyme-bridging model for DNA relaxation. The crystal structure of the 67kDa fragment of E.coli enzyme suggests how such reaction occurs. The active site tyr319 is buried in the structure. It is proposed that as the single stranded DNA binds to the cleft, domain III undergoes conformational adjustment to place the nucleophilic O-4 oxygen of the tyrosine side chain in a position to attack the phosphate. After the cleavage the active site tyr319 is covalently bound to the 5’ phosphate on one end of the cleaved and the other end is proposed to occupy a nucleotide binding site at the end of cleft in
Str-3
Immediately upon cleavage domain III that is holding onto the 5’ end of the broken strand, lifts away from domain I to create a gap through which is passed either intact strands. Once the intact strands have moved into the hole of the torus, the clamp closes and the cleaved strand is religated. The protein must then opened and closed second time to release the passed strand to complete the cycle. Once reset, the enzyme can dissociate from the DNA or act processively to carry out another cycle of strand passage.
Since this series of reactions occurs without an external energy source such as ATP.
TYPE II DNA TOPOISOMERASES
The following properties are shared by all type II topoisomerases:
- The dimeric enzyme binds duplex DNA and cleaves the opposing strand with a four base stagger ( topo VI may generate two base stagger).
- Cleavage involves covalent attachment of each subunit of the dimmer to the 5’end of the DNA through a phosphotyrosine bond.
- A conformational change pulls the two ends of the cleaved duplex DNA apart to create an opening in what is referred to as gated or G-segmented DNA. A second region of duplex DNA is referred to as transported or T-segment is passed through the open DNA gate.
- The reaction requires Mg and ATP hydrolysis is required for enzyme turnover and rapid kinetics.
Str-4
The positively charged central groove that run across the dimmer interphase possesses the correct dimensions to function as the binding site tyrosines are juxtaposed with the promoters at the dimmer interface would be expects to create a DNA gate for the transport reaction.
The homologous yeast spo11 protein is involved in producing double strand breaks that initiate the process of meiotic recombination, but unlike a topoisomerase,the enzyme dose not rejoin the two ends and is instead cleaved away from the DNA thus, the spo11protein functionally behave like a type II topoisomerase that is unable to close the gate after cleavage. One possible explanation for the properties of spo11is that spo11 lacks a functional analogue of the topoisomerase VIB subunit bridge and cannot prevent dissociation of the A subunit. If the above hypothesis is correct, then is seems likely that association with one regulates the cleavage activity of spo11 or more accessory proteins, none of which provide abridge between the two A subunits. Any combination of the nine other proteins required in yeast for producing double-strand breaks during meiosis could function in this regard.
GENERAL
The molecular techniques such as plasmid isolation, restriction digestion, ligation, bacterial transformation etc. were based on protocols described by Sam brook et.al. (2001).
Stock solutions of various reagents were prepared in RO water. Stocks of various antibiotics were prepared in DMSO and stored at -20 ºc. Water needed for PCR reactions, restriction digestion, precipitation of DNA etc were obtained from Elix™, Millipore water purification systems, France.
All aseptic procedures were carried out under sterilized conditions in laminar flow hoods. (Kartos international, India)
CHEMICAL REAGENTS AND GLASSWARES USED
General use chemicals were of AR grade procured from Qualigens, Difco Hi media (India). Antibiotics and other fine chemicals were purchased either from Sigma Chemical Co. St. Louis, USA. Nitrocellulose paper (Hydrogen C), and Mega prime labeling kit were obtained from Amersham, USA. Restriction enzymes were obtained either from Roche Molecular Biochemicals, Germany or MBI Fermentas. USA. All the glass wares used was purchased from Borosil, India. Plastic wares such as micro tips, micro centrifuge tubes, Petri plates etc. were obtained from Tarson, India or Axygen USA.
RICE GENOMIC DNA ISOLATION
Total DNA was extracted from plant tissue following the procedure of Dellaporta et.al. (1983) with slight modifications. Around 3 gm of shoot portion was taken from 4 days old etiolated ice seedlings, so as to minimize chloroplast and mitochondrial DNA contamination. The tissue was ground to fine powder in liquid nitrogen using precooled mortar and pestle with the help of liquid nitrogen .The powdered tissue was transferred to precooled SS-34 tube and 15 ml of extraction buffer (100Mm Tris –Cl, pH 8.0, 50.0mM EDTA,PH 8 ,500Mm NaCl and 10mM B-ME)added immediately . The tissue was homogenized by vortexing followed by the addition of 1 ml of 20% SDS. The tubes were kept at 65ºc for 20min. with intermittent vigorous shaking. Subsequently, 5.0ml of 5M potassium acetate was added and the tube incubated on ice for 30min. After vigorous shaking. The tube was then centrifuged at 15,000 rpm for 20min at 4ºc in a centrifuge. The supernatant was carefully transferred to a fresh SS-34 tube through two layers of autoclaved muslin cloth and 10ml isopropanol added, properly mixed and incubation carried out at -70 ºC for 1 hr. The DNA was pelleted by centrifugation at 12,000 rpm for 15 min at 4ºC in Sorvall SS-34 rotor. The pellet was suspended in 0.7 ml of high salt buffer (50 mM Tris-Cl, pH 8.0 and 10 mM EDTA, pH 8.0) and kept for dissolving in ice for 10-15 min. This step onwards wide bore tips (1 ml cut tips) were used for pipeting the genomic DNA in order to avoid shearing. The liquid was transferred to a microfuge tube and centrifuged at 13,000 rpm for 10 min at 4ºC. The supernatant was transferred to a fresh microfuge tube and undissolved debris discarded. 6µl of RNase (10µg/µl) was added and incubated at 37ºC for 1 hour. Subsequently, 350µl of phenol and 350µl of chloroform was added, mixed properly and then centrifuged at 10,000rpm, 4ºC for 10 minutes. The aqueous phase was taken in fresh MCT and 700µl of chloroform was added, mixed gently and centrifuged at 10,000rpm, 4ºC for 10 minutes. Aqueous phase was taken and 1/10th volume of 3M sodium acetate, pH 5.2, and 0.7 volume of isopropanol were added and mixed gently to precipitate the DNA and kept at room temperatures. The DNA was pelleted at 10,000rpm for min, washed with 70% ethanol and dried in speed Vac TM (Savant Instruments Inc., NY). The pellet was dissolved in minimum volume of 1X TE buffer, PH 8.0.
DNA QUANTIFICATION
The DNA was quantified spectrophotometrically (DU640B spectrophotometer, Beckman Instruments, USA) in a quartz cuvette by measuring absorbance of 1µl aliquot, diluted with 1ml with RO water, take O.D at 260nm to quantitate the DNA. And also take O.D at 280nm to qualitate the DNA.check the DAN quality by loading on 0.8% agarose gel.
Absence of smear below the band was taken as the criteria for selecting high molecular weight DNA.
POLYMERASE CHAIN REACTION
The polymerase chain reaction can also be used to obtain a pure sample of a gene. This is because the region of the starting DNA molecule that is copied during PCR is the segment whose boundaries are marked by the annealing positions of the two oligonucleotide primers if the primers anneal either side of the gene of interest, many copies of that gene will be synthesized.
In the PCR amplification was carried out using Gene Amp. PCR system 2400 (Applied Biosystems Switzerland)
The gene specific primer sequences used for PCR reaction are as follows:
PCR amplification of the spo11 gene was done at low annealing temperature. PCR amplification was carried out in the following conditions;
For the low annealing temperature PCR:
Denaturation temperature was 94ºC for30 sec. annealing temp. 65ºC for 30 secs.and 70ºC, and followed by extension at 70ºC for7 mins.
DNA = 1µl
5’ prime (forward prime) = 1µl
3’ (reverse prime) = 1µl
dNTP = 1µl
Buffer = 5µl
MgCl2 = 4µl
Taq polymerase enzyme = 1µl
MQW = 36µl
Total reaction volume = 50µl
The mixture was heated to 94ºC at which temperature the hydrogen bonds that hold together the two strands of the double stranded DNA molecule are broken, causing the molecule to denature.
The mixture is cooled down to 60ºC to 65ºC .The two strands of each molecule could join back together at this temperature, but most don’t because the mixture contains a large excess of short DNA molecules, called oligonucleotides or primers, which anneal to the DNA molecules at specific position.
The temperature was raised to 70ºC this is the optimal working for the Taq DNA polymerase that is present in the mixture that it attaches to one end of the each primer and synthesize new strands of DNA, complementary to the template DNA molecules, during this step of the PCR .Now we have four strands of DNA instead of two that they were to start with.
The temperature is increased back to 94ºC the double stranded DNA molecule, each of which consists of one strand of the original molecule and one new strand of DNA, denature into single strands. This begins a second cycle of denaturation –annealing –synthesis, at the end of which there are eight DNA strands. By repeating the cycle in this cash 35 times the double stranded DNA molecule that we begin with is converted in to over 50 million new double stranded molecules, each one a copy of the region of the starting molecule (SPO11 GENE) delineated by the annealing sites of the two primers. The amplified product was then resolved on a 1% agarose gel.
PURIFICATION OF PCR PRODUCT
QIA Quick Gel extraction Kit protocol. (70 bp to 10 kb)
Load the amplified product on the 0.9% agarose gel along with λ marker
Cut /excise the DNA fragment from the agarose gel with a clean sharp scalper
Weigh the gel slice in a colorless tube. Add 3 volume of buffer QG to 1 volume of the gel (100mg ~100µl)
Incubate at 50ºc for 10 min. (or until the gel slice has completely dissolved). To help dissolve the gel, Mix by vortexing the tube every 2-3min. during the incubation
After the gel slice has completely dissolved, check that the color of the mixture is yellow. (Similar to buffer QG without dissolved agarose)
If the color of the mixture is orange /violet add 10µl of sodium acetate, pH
5.0and mix the color will turn yellow
[The adsorption of the DNA to QIA quick membrane is efficient only at pH
≤7.5. Buffer QG contains a pH indicator, which is yellow at pH< 7.5 and orange or violet at higher pH, allowing easy determination of the optimal pH for DNA binding]
Add 1 gel volume of isopropanol to the sample and mix (If agarose gel slice is 100mg, add 100µl of isopropanol. This step increases the yield of DNA fragment <500bps.and >4kb. For DNA fragments between 500bp and 4 kb addition of isopropanol has no effect on the yield .Don’t centrifuge at this stage)
Place a QIA quick spin column in a provided 2 ml collection tube
To Bind DNA, apply the sample to QIA quick column, and centrifuge for 5 min.
Discard the flow through and place QIA quick column back in the same collection tube (collection tube can be reused).
OPTIONAL: Add 0.5 ml of buffer QG to QIA quick column and centrifuge for 1 min. (this step removes all traces agarose. It is required when the DNA will be used for direct sequencing, in vitro transcription or micro injection).
To wash add 0.75 ml of buffer PE to QIA quick column and centrifuge for 1 min (wait for 5 min. centrifugation)
Discard the flow through and centrifuge the column for an additional one min. and place the column into a clean 1.5 ml microfuge tube
To elute DNA, add 50µl of buffer EB (10m M TrisCl Ph8.5), to the centre of the column and was shaken then spinned for 1 min.at maximum speed...
CLONING OF THE SPO11 GENE
The basic steps in gene cloning experiment are as follows:
- A fragment of DNA, containing the gene to be cloned, is inserted in to a circular DNA molecule called a vector. To produce a chimera or recombinant DNA molecule.
- The vector acts as a vehicle that transports the gene in to a host cell, which is usually a bacterium; although other types of living cells can be used.
- Within the host cell the vector multiplies, producing numerous identical copies not only of itself but also of the gene that it carries.
4. When the host cell divides copies of the recombinant DNA molecule are passed to the progeny and further vector replication takes place.
5. After a large number of cell divisions, a colony or clone, of identical host cells is produced .Each cell in the clone contains one or more copies of the recombinant DNA molecule; the gene carried by the recombinant molecule is said to be cloned.
LIGATION WITH VECTOR
The vector used in this experiment was Pbsk (+), which has the restriction sites for both restriction enzymes, Xba I and Sac I.The enzymes in both the vector and the insert produce a complimentary sticky ends.
The PCR amplified product was precipitated from the reaction mixture and digested with Xba I and Sac I (Roche Molecular Biochemical’s, Germany) as per manufacturer’s specifications. The digested product was purified by phenolization followed by ethanol precipitation. The vector PBSK (+) was digested with the same set of enzymes and purified in the same fashion. For cloning, the digested vector and PCR product were mixed in the ratio of 1:10 and ligation carried out in total reaction volume of 15µl using 1.5 units of T4DNA Ligase (Roche Molecular Biochemical’s, Germany) as per manufacturer’s specification at 16ºc for at least 16 hrs. The ligated product was then transferred to the competent cells and the recombinant clones were selected and the plasmid was isolated by minipreparetions kit for further manipulations.
PREPARATION OF COMPETENT HOST BACTERIA (XL-1 BLUE MRF’) CELLS
For cloning of the Spo11 gene, host bacteria (E.Coli XL-1Blue MRF’) were made competent .Host bacteria were grown overnight in 250ml of LB( luria – Bertani) medium (1% NaCl ,1%Bacto tryptone ,0.5 % yeast extract) in the presence of 0.2% maltose, 12.5μg/ml tetracycline antibiotics and, incubated at 37ºc and 200rpm.in an incubator shaker.
200µl of the over grown E.coli XL-1 Blue MRF’ cells were taken in fresh 25ml of LB medium and were incubated at 37ºc, 200rpm for1 to 1 and half hour till an OD260 of 0.5 was obtained .The bacteria culture was pelleted at 4000 rpm for 10 min in autoclaved centrifuge tube (SS 34,Sorvall RC5B) and the pellet was gently resuspended in 5 ml of 0.1M MgCl2 incubate in ice for 20 to30 min then a spin was given at 4000rpm, at 4ºc for 10 min.The supernatant was decanted and to the pellet 1 ml of 0.1M CaCl2 was added and incubated in ice for 10 min.After 10 min ice incubation a spin was given at 4000 rpm, at 4ºc for 15 min. Finally 300ul of 0.1M CaCl2 was added to the pellet and incubate in ice for 2hr to be used for transformation.
TRANSFORMATION OF BACTERIA
Competent bacterial host cells (E .coli strain XLI-Blue MRF`, 100µl) were mixed with 5 to 10µl of ligated samples and incubated on ice for 30min.with gentle shaking, after 10min. The mixture was subjected to 5 alternate heats (42ºc, 35 sec) and cold (on ice 45 min.) shocks and incubated on ice for 10 min. Subsequently, 1 ml LB medium was added to the mixture and incubated at 37ºC for 2 hr. to allow recovery of the bacteria and expression of antibiotic resistant encoded by plasmid. The cells were plated on 1.5% LB agar , TAXI plate containing 40mg/ml X-Gal (5-bromo -4 –chloro-3- indoly- B – D –galactoside), 0.2mM IPTG ,12.5 µg/ml tetracycline and 50ug/ml ampiciline and incubated overnight in an inverted position at 37ºc .
SELECTION CLONES OF THE RECOMBINANT
The bacterial colonies harboring recombinant plasmids were differentiated by their white color from those harboring non-recombinant plasmids appeared blue in color. The white colonies were patched on to fresh plates containing appropriate antibiotics. Pick white colony with autoclaved toothpick patch on TAXI plate and also inoculate in LB broth containing antibiotic. Those retaining their white color after 16to 20 hr of incubation at 37ºc were used for plasmid isolation.
PLASMID DNA ISOLATION
Plasmid DNA was isolated following the alkaline lyses protocol (Biraboin and Doly, 1979)
This procedure was employed for the isolation of the plasmid DNA harboring the cloned gene. This method involves use of three different solutions that have the following composition:
Solution I;
10mM EDTA, pH8.0
25Mm tris-Cl, PH 8.0
50mM Glucose
Solution II;
0.2N NaOH
0.1%SDS
Solution III;
3.0M potassium acetate
1.8M formic acid
Bacterial cultures were initiated from single colonies in small glass vials containing 5 ml LB broth with ampicilline(50µg/ml) and tetracycline (12.5µg/ml) and grown at 37ºC over night at 200rpm. The culture was pelleted at 5000rpm at 4ºC for 5 min. And the pellet was resuspended in 100µl ice cold solution І, by vortexing and incubating on ice for 5 min. .Then 150µl of solution II was added and mixed thoroughly but gently by inverting the tube several times followed by incubation at room temperature for 5 min. Then 200μl Solution III was added and mixed well followed by incubation in ice for 10 min. The mixture was centrifuged at 13,000rpm, at 4ºC for 10 min,and the supernatant was phenolized with equal volume of phenol: chloroform :isoamylalcohol (25:24:1) ,and then with chloroform :isoamyalchol (24:1) by centrifugation at 13,000rpm, 4ºC, for15min. The supernatant was transferred to fresh eppndorfs tubes and precipitated with 2 volume of isopropanol and centrifuged at 13,000rpm at 4ºC for 15 min. The pellet was washed twice with 70% ethanol, dried and then dissolved in 15-20µl of T.E buffer.
ANALYSIS OF HOMOLOGY OF THE SPO11 IN RICE
The genomic DNA isolated from rice steam by the dellaporta method was subjected to restriction digestion in 80µl of reaction volume with ECO RI, XhoI, BamhI, Hind III, XbaI and ECORV as per the manufacturer specification.
Mini gel for southern blotting was prepared (Southern, 1975). Agarose to final concentration of 1% was added to 150ml of 1x TAE buffer and boiled for dissolving. After the agarose solution attained a moderate temperature, by putting the flask under running tap water, EtBr solution was added to a final concentration of 0.5µg/ml. The agarose solution was poured in to a gel tray and allowed to polymerize for 30-45 min. The sample was loaded along with standard size marker (10kb ladder of DNA) and also undigested sample of the rice genomic DNA was loaded along the marker. The gel was run overnight at constant voltage (35V).place the gel on U.V transilluminator and photographs it using Polaroid camera along with fluorescent scale. To facilitate the transfer of electrophoreses DNA on to the membrane the gel was subjected to the following treatments.
Depurination: The gel was safely transferred to tray and sufficient amount of 0.15N HCl was added; so as to completely submerge the gel. The tray was left on the shaker for 15 min. after which the solution was discarded and the gel rinsed with Milli Q water.
Denaturation : Appropriate amount of denaturation solution (4M NaCl and 5 M NaOH) was added to the tray. The gel was incubated in the solution for 45 min. while on a shaker. After the treatment the solution was discarded and the gel rinsed with MQ water.
Neutralization: Finally the gel was incubated for 45min. in neutralization solution (2M Tris-HCl, PH8.0 and 4M NaCl) before DNA transfers.
Transfer –In a tray 20x SSC solution was taken and a glass plate over it .one strip of the Whitman’s paper were used as a wick. On the top of the wick two sheets of the Whitman’s paper 15cmx13cm were placed and wetted with 20XSSC without air bubbles.
Gel is then carefully inverted over the Whitman’s paper and the left hand of the gel was marked. On the top of the gel , Nylons membrane 15x13cm prewetted in RO was placed and briefly wetted in 20x SSC .
On the top the membrane two more Whitman sheets 15x13cm. Were placed followed by stack of blotting sheets. At the top of the glass sheet a suitable weight was placed to facilitate blotting. Blotting was allowed to continue overnight after which the nylon membrane was picked and air dried for 30 min. The nucleic acid were UV cross linked on to the membrane (Amersham, international Inc., UK)
Prehybridization and hybridization was then done.
The Radioactive probe was prepared using mega prime labeling kit and hybridized was carried out over night at 42ºc.
PROBE PREPARATION
Cast a 1.2% low melting point agarose gel, and then load the PCR product in the well. Cut the specific band from the gel, then melt the gel and denature the DNA in the boiling water bath, took 21µl of this and add 5μl of primer solution and boiled for 5 min, then cool at 37ºC, then add the following components in the following order:
Buffer (10x) = 5µl
dTTP = 4µl
dGTP = 4µl
dCTP = 4µl
α32 p ATP = 5µl (done in the radioactivity room)
Enzyme (klenew) = 2µl (done in the radioactivity room)
Mixed gently and incubate the mixture at 37ºc for 1 hr and boil for 5 min, then add this probe in hybridization solution, then incubate with the blot in hybridization solution for 16hr at 42οC
Washing of blots and Exposure
Preparation of washing solution
Solution; I 5X SSCsolution with 0.1% SDS
Solution; II 2X SSCsolution with 0.1% SDS
Solution; III 1X SSCsolution with 0.1% SDS
The blot was washed with washing solution I, II and III until the radioactive detector reading is <5, then wrapped the blot in saron wrap mark the two side of blot with radioactive day spots for orientation identification, then place the X-ray film against the blots in the hyper castes in the darkroom, then place the X-ray film in -80οC for 3-4 days
CLONING OF SPO11 DNA IN pCTB VECTOR
The pbsk (+) recombinant plasmid were isolated and digestion was carried out as describe earlier. The insert DNA were extracted by QIA gel extraction kit. After following extraction, vector pCTB were digested with Sac I and Xba I according to the manufactures specifications since the two enzymes have compatible buffers (buffer A) digestion by both enzymes were set up simultaneously.
Composition of digestion mixture was as follows;
Digestion was carried out overnight at 370C and precipitated after phenol digestion as described earlier.
LIGATION OF DNA WITH pCTB VECTOR
Restricted vector and gel eluted DNA samples were mixed in 1: 10 ratio and ligation was carried out in 15µl reaction volume, using T4 DNA ligase.
Composition of ligation mixture was as follows;
Ligation was carried out overnight at 160C
TRANSFORMATION AND PLATING OF BACTERIA
E.coli strains (XL1Blue MRF’) were made competent and transformed with ligated product according to CaCl2 method as described earlier. Transformed bacterial cells were plated on LB medium containing 25μg/ml kanamycin and 12.5μg/ml tetracycline, solidified with 1.5% agar. After 30 minutes of plating the plates were incubated overnight at 370C.
SELECTION OF RECOMBINANT CLONES
.
Only transformed bacterial cells produce colonies because of antibiotic effect some selected colonies were patched and inoculated on fresh LB agar plate and LB broth, both containing 50μg/ml kanamycin and 12.5μg/ml tetracycline. Incubate it for 16hr at 37οC.
PLASMID ISOLTON AND GEL ELECTROPHORESIS
Bacterial cultures were initiated from selected colonies in 1.5 ml LB broth with 25µg/ml kanamycin and 12.5μg/ml tetracycline. Allowed to grow at 370C, 200rpm.
The culture were pelleted at 5000rpm at 40C for 5 minutes, plasmids were isolated by alkaline lysis protocol given by Biraboing and Dolly as described earlier.
Isolated plasmids were run on 1% agarose gel using 1X TAE buffer. Plasmid DNA were viewed on UV Transilluminator and photographed in gel documentation unit.
RESTRICTION ANALYSIS OF RECOMBINANT PLASMID
Plasmid sampled isolated by miniperparation method were digested in 30µl reaction volume with Xba I and Sac I under conditions specified by the manufactures specifications, incubated it for 5hr.The digested samples were loaded on 1% agarose gel along with standard size marker (10kb DNA ladder) and observed on a UV transilluminator for selection of the recombinant clones containing the fragment of desired size.
TRANSFORMAION OF RECOMBINANT VECTOR IN AGROBACTERIUM TUMEFACIENCE
Inoculate a single colony of A.tumefaciens in 30ml of YEM broth containing rifampicin (100μg/ml) grow this culture for 3 days at 28°c on 200rpm, then subculture 2ml of primary inoculums in to 30ml of same broth incubate it till the 0.6 O.D was obtain at 260nm. Then spin the culture at 4000rpm 15min at 4°C, then resuspend the pellet in ice-cold 10mM CaCl2, (to make the cells competent) and then take 100μl of bacterial cells and 1μl of pCTB recombinant plasmid (isolated by QIA mini plasmid isolation kit) Incubate at 37°c for 5min then add 700μl of YEM broth incubate it over night at 28°c on 200rpm. After incubation bacterial cells were plated on YEM medium containing 100μg/ml rifampicin and 50μg/ml kanamycin. After 30min of plating the plate were incubated for 2days in dark at 28°c
SELECTION OF RECOMBINANT CLONES
Only bacterial colonies harboring recombinant plasmid were growing in a medium because of antibiotic effect. The selected colony was patched and inoculated in fresh YEMagar plate and YEM broth respectively both contain rifampicin 100μg/ml and kanamycin 50μg/ml .Incubate it at 28°c for 2 days .
PLASMID ISOLATION FROM AGROBACTERIUM TUMEFACIENS
The culture was pelleted at 5000rpm at 4°c for 10min plasmid was isolated by alkaline-lysis method as describe earlier with some modification like we added 20μl of 20mg/ml lysozyme. Isolated plasmids were run on 1% gel using 1XTAE buffer. Plasmid DNA were viewed on a U.V transillminator and photographed in gel documentation unit.
After isolation of plasmid from A.tumefaciens the same plasmids were transformed into E.coli XL1 MRF’ competent cells
And again transformation, plasmid isolation and restriction digestion done as describe earlier.
To recheck correct transformation of recombinant plasmid in A.tumefaciens