Leaf mesophyll protoplasts were prepared from B73 maize seedlings as described by Sheen et al, (http://molbio.mgh.harvard.edu/sheenweb/protocols_reg.html ). DNA was purified using the Plasmid Miniprep Kit from Sigma and adjusted at a concentration of 1 µg/µl for transfection into protoplasts. After transformation, the protoplasts were incubated overnight. TM (5 µg/ml) treatment was carried out the next morning for 3 and 6 hr. Untreated protoplasts and protoplasts TM treated for 3 and 6 hr were visualized for SGs using a Leica SP5X MP confocal laser scanning microscope with a 63X oil immersion objective lens and excitation and emission wavelengths set at 520 and 550 nm for YFP.
Plasmid miniprep kit
Manufactured by Merck Group
Sourced in United States, Sao Tome and Principe, Germany
The Plasmid Miniprep Kit is a laboratory tool designed for the rapid and efficient extraction and purification of plasmid DNA from bacterial cultures. The kit utilizes a standard alkaline lysis method followed by column-based purification to provide high-quality plasmid DNA suitable for downstream applications such as cloning, sequencing, and transfection.
Automatically generated - may contain errors
Lab products found in correlation
Zymolyase, by G Biosciences (1 mentions)
Plasmid miniprep kit, by Qiagen (1 mentions)
Instaclone pcr cloning kit, by Thermo Fisher Scientific (1 mentions)
Acid washed glass beads, by Merck Group (1 mentions)
Calf intestinal alkaline phosphatase, by New England Biolabs (1 mentions)
Prsetb, by Thermo Fisher Scientific (1 mentions)
Nickel nta beads, by Qiagen (1 mentions)
Plasmid midiprep kit, by Qiagen (1 mentions)
Anti his monoclonal antibody, by Merck Group (1 mentions)
Dh10bac, by Thermo Fisher Scientific (1 mentions)
Pgem t easy, by Thermo Fisher Scientific (1 mentions)
Pgem 3z, by Thermo Fisher Scientific (1 mentions)
Dab 3 3 diaminobenzidine, by Merck Group (1 mentions)
Ncoi and hindiii, by New England Biolabs (1 mentions)
Pfastbachtb, by Thermo Fisher Scientific (1 mentions)
Pd 10 column, by GE Healthcare (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Trizol, by Merck Group (1 mentions)
Ampicillin sodium salt, by Merck Group (1 mentions)
Pmal c5e, by New England Biolabs (1 mentions)
7 protocols using plasmid miniprep kit
1
Visualizing Stress Granules in Maize Protoplasts
2
Isolation and Identification of Yeast Plasmid Suppressors
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Yeast cells harvested from 25 ml saturated culture of sorted libraries were resuspended in buffer P1 (supplied with Qiagen plasmid miniprep kit) and vortexed in the presence of acid-washed glass beads (SIGMA) for 10 min. The suspension was incubated with Zymolyase (30U, G-Biosciences) at 37oC for 4 hr to break the cell wall. A Qiagen plasmid miniprep kit was used for further downstream processing of the cells to purify the plasmid. CcdB gene inserts amplified from the libraries were cloned into the pTZ57R/T TA vector using an InsTAclone PCR cloning kit (ThermoScientific) and transformed into E. coli XL1-Blue cells for blue-white screening (Langley et al., 1975 (link)). The CcdB inserts from 96 randomly picked white colonies derived from each library after the final round of sorting, were sequenced by Sanger sequencing at Macrogen, Korea, to identify second-site suppressors.
3
Recombinant Protein Expression in E. coli and Insect Cells
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Strains used for the propagation of the plasmid DNA—E. coli DH5α, DH10BAC, for the expression of recombinant proteins cells—E. coli BL21 (DE3), insect cell line Sf21 and the cloning vectors pRSET-B, pFASTBAC-HTB, pGEM-3Z and pGEM–T Easy were procured from Invitrogen, USA. The restriction enzymes KpnI, NcoI and HindIII, Calf-Intestinal Alkaline Phosphatase, Pfu and Taq polymerases were obtained from New England Biolabs, USA. ATP and T7 RNA polymerase were purchased from Fermentas, USA. Nickel-NTA beads, DEAE-cellulose columns and plasmid midi-prep kit were procured from Qiagen, USA. PD-10 columns were purchased from GE Healthcare Life Sciences, USA. Trizol, DAB (3,3′-diaminobenzidine), Lysozyme, anti-His monoclonal antibody, plasmid miniprep kit and protease inhibitor cocktail were purchased from Sigma Chemicals, USA. Isopropyl β-d -1-thiogalactopyranoside (IPTG) was acquired from GIBCO-URL, USA. An antibody was raised in rabbit earlier against recombinant PPRV L protein domain 3 (1717–2183 a.a) expressed in E. coli [3 (link)]. γ-P32-ATP and α-P32-ATP were purchased from BRIT, Mumbai, India. The oligonucleotides were supplied by Sigma Chemical Co., India and were used to generate in vitro transcribed RNA substrate for RTPase assays.
4
Bacterial Expression System for Mutagenesis
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Escherichia coli DH5α (New England Biolabs (NEB), Ipswich, MA, U.S.A.), pMAL c5E (NEB), Plasmid miniprep kit (Sigma chemicals, St. Louis, MO, U.S.A.), Quick-change site-directed mutagenesis kit (Stratagene, Cedar Creek, TX, U.S.A.), Ampicillin sodium salt (Sigma), Primers (Sigma), DpnI enzyme (Fermentas, Waltham, Massachusetts, United States), IPTG (Sigma), Luria-Bertani (LB) broth (Himedia, Mumbai, India).
5
Cloning of COL7A1 Minigene Construct
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Screening constructs were designed and cloned according to the protocol of Bauer et al. [12 (link)]. For cloning of the target region intron 102/exon 103 of COL7A1 into the COL7A1-minigene (COL7A1-MG) expression vector an intron 102 specific forward primer (5'-GATCGATATCACAGGGAAGAGGGTGGTGTACTCTC-3'), an exon 103 specific reverse primer (5'-GATCGCGGCCGCCTGGGGTCCCAGGAGTCCACG-3') and genomic DNA from a healthy donor were included in the PCR. The PCR product was cloned into the screening vector using the restriction sites for EcoRV and NotI. Gel-extractions of amplified PCR products were performed using a GFX™ PCR DNA and Gel Band Purification Kit (GE Healthcare, Chalfont St. Giles, UK). Plasmid preparations were carried out using a Plasmid Mini Prep Kit (Sigma–Aldrich, Taufkirchen, Germany), according to the manufacturer’s protocol. Sequence analysis of all plasmids and PCR products was performed using a 3500 ABI automated sequence analyzer and ABI PRISM dye terminator cycle sequencing kit (Applied Biosystems, Foster City, CA, USA).
6
Cloning and Characterization of YcjR
7
Conjugation Assays for Plasmid Transfer
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Conjugation assays were performed as described (21 (link)) using UB1637 (SmR) (22 (link)) as recipient cell and DH5α containing relevant plasmids as donor cells. Conjugation followed the same conditions as reported, using 1 h as conjugation time (20 (link)). After conjugation, transconjugants were pooled together, DNA extracted using a plasmid mini-prep kit (Sigma-Aldrich) and their nic-sites sequenced and compared with the electropherogram of the sequence of the pool of donors. A scheme of the experimental process is presented in Figure 2 . In the case of the partially randomized libraries, isolated transconjugants were selected after conjugation and their DNA was extracted. nic-sites were sequenced and the different variants found were characterized in terms of conjugation frequency. DNA was extracted and used to transform new donors to obtain individual conjugation frequencies. Each experiment was repeated six times. Conjugation frequency was calculated as the number of transconjugants divided by the number of donor cells.
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