Comet assay to study DNA damage. Read more
Authors: Sandra Sernbo
+1
+1
-1
To infect suspension cell lines with lentiviral particles. Read more
Authors: Sandra Sernbo
+1
+1
-1
The following protocol is the standard procedure by which the in situ PLA service is performed at the PLA proteomics facility: http://www.scilifelab.se/facilities/pla-proteomics/ Depending on sample type (cells, FFPE tissue, fresh frozen tissue) the preparation before PLA analysis varies in terms of fixation, permeabilization, etc, which must be optimized for each sample type. The PLA protocol described here is the same for all sample types. The reaction volume is 40 µl per 1 cm2 sample area. All incubations are performed in moisture chamber to avoid drying of samples. Read more
Authors: Masood Kamali-Moghaddam
+1
0
-1
KI Biobank is a core facility in with the mission of supporting researchers with high quality biobank services at Karolinska Institutet. This protocol regulate the manual DNA extraction at the KI Biobank and was developed in accordance with the requirements of an accredited facility Read more
Authors: KI Biobank
+1
+1
-1
Lepidium campestre has proven to be one of the more difficult plant species for DNA isolation. Here we describe a method that has proven to produce good and clean DNA for RAD sequencing purposes. There are several stages to the protocol. First the leaf samples are pulverized and proteins are broken down. Second stage is the actual extraction of DNA which should follow directly after the pulverization and protein breakdown. Third stage comprises of quality control and RNA removal. This is essential if the samples are intended for example RAD sequencing. The fourth and final stage of the process is to estimate the DNA concentration. This is not as straightforward as it may sound, because L. campestre appears to have long strands of genomic DNA coiling and clumping together. If this type of clumping and uneven distribution of DNA happens in the solution, for example Nanodrop will not provide accurate estimates of DNA concentrations. We describe a way to relax the DNA and make it possible to obtain more accurate estimates of DNA concentrations. Read more
Authors: Fernando Lopes Pinto and Tytti Vanhala
+1
0
-1
Lepidium campestre has proven to be one of the more difficult plant species for DNA isolation. Here we describe a method that has proven to produce good and clean DNA for RAD sequencing purposes. There are several stages to the protocol. First the leaf samples are pulverized and proteins are broken down. Second stage is the actual extraction of DNA which should follow directly after the pulverization and protein breakdown. Third stage comprises of quality control and RNA removal. This is essential if the samples are intended for example RAD sequencing. The fourth and final stage of the process is to estimate the DNA concentration. This is not as straightforward as it may sound, because L. campestre appears to have long strands of genomic DNA coiling and clumping together. If this type of clumping and uneven distribution of DNA happens in the solution, for example Nanodrop will not provide accurate estimates of DNA concentrations. We describe a way to relax the DNA and make it possible to obtain more accurate estimates of DNA concentrations. Read more
Authors: Fernando Lopes Pinto and Tytti Vanhala
+1
+1
-1
This protocol describes the workflow for manual DNA extraction at the KI Biobank. Read more
Authors: KI Biobank
+1
0
-1
Biotinylation of an antigen is often the method of choice for protein immobilization to select and evaluate affinity reagents. The tight and specific interaction of biotin with streptavidin or avidin is thereby used in many selection systems to specifically capture the antigen and/or affinity reagent for further analysis. In vitro biotinylation is frequently used, whereby lysine residues in the antigen are chemically modified. Biotinylation of a short acceptor peptide in vivo is an attractive method to achieve site-specific modification without the risk to interfere with protein folding or function of the antigen. In vivo biotinylation is achieved by co-expressing the protein of choice (fused to a biotin acceptor peptide) and the bacterial biotin-protein ligase, holocarboxylase synthetase (BirA) in the presence of biotin. Read more
Authors: Tracy Keates
+1
+2
-1
Chemoselective-Easy to Perform-Versatile-High Yielding. Click chemistry for antibody-DNA conjugation reaction is a very safe reaction because DBCO and azides are both are absent from biological systems, so this safe chemistry produce minimal background for bio-analysis compare others chemistry. Other hand the copper free click reaction condition is very suitable for biomolecules and also easily can to perform this conjugation with general laboratory skill. Ultimately the reaction having advantages; very specific alkyl and azide reaction produce highly stable trioazole bond between antibody-DNA. This click conjugation conjugate probe is very stable for long time storage at 4°c compare others chemistry. Here is the summary of our current click chemistry protocol for the lab: 30 μl antibodies (2μg/μl in PBS) were activated with a 20-fold molar excess 2μl 4mM of the cross linker Dibenzylcyclooctyne-NHS ester (CLK-A102N, DBCO-NHS ester) dissolved in DMSO incubates at room temperature for 30 min. The reactions was stopped by adding 3μl 1M Tris-Hcl, pH 8.0. Incubate at RT for 5 min. The excess DBCO-NHS ester was removed from the activated antibody with the equilibrated Zeba Spin Desalting Column (7k MWCO, Thermo Scientific). After purification, The DBCO-labeled antibodies were mixed with 4-fold molar excess azide-modified Oligonuclotides (100μM) incubated overnight at 4°C. Read more
Authors: Rasel A. Al-Amin and Ulf Landegren
+1
+1
-1
There have also more challenges for maleimide chemistry to breakdown of di-sulfate bond of heavy chain of antibody in the protocol for antibody-DNA conjugation. Malemide/NHS-esther (Sulfo-SMCC) chemistry; the maleimide moiety itself is very unstable in solvent and overall very poor stability of conjugate for long-term storage. Sulfo-SMCC chemistry DNA based technique for bio-analysis generates more background in biological environment compare others chemistry like Sulfo-SANH chemistry and click chemistry. Summary: Antibody concentrate: Amicon Ultra-10K column (millipore), 15°c, 14000g for 30min. Collection: 1000g, 2 min. Activating antibody: To 10 μl antibody in 1x PBS (2 µg/µl) add 1 μl 4mM sulfo-SMCC (MW: 436.37, 0.45mg of SMCC in 257.81μl DMSO for 4mM) freshly dissolved in DMSO (~30X excess over Ab), mix well by tapping the tube/vortexing and incubate at RT for 2 hrs. Reducing oligo: Degas the oligo by heating at 95°C for 3 min, and then directly put on ice for 1,5 min. 3 µl (100 µM) oligo for each 10 µl (20 µg) Ab. Reduce the oligo by mixing 25 mM of 1.2 µl DTT (DL-Dithiolthritol: 154.25 MW, final 50 mM, 1 mg of DTT dissolved in 259.32µl 1xPBS-5mM EDTA ) with 3 µl oligo (final 20µM), so molar ratio DTT/oligo 100:1. Incubate for 1h at 37°C (water bath). Zeba 7k Desalting Column conditioning – Equilibrate the columns by adding 300 µl of 1X PBS-5 mM EDTA. Spin 3000 rpm, 1', RT. Discard the eluate and repeat the equilibration step twice. Purifications (excess SMCC and DTT): Add up the volume of the antibody/oligo to minimum 30 µl. Place the antibody/oligo in the Zeba spin column. Let it absorb. Add 15 µl 1xPBS-EDTA to the column. Spin at 3000 rpm, 2', RT and eluted. Conjugation reaction: Mix the antibody with the oligo. Mini Dialyse 7KD, Thermo Scientific against in 1 lit 1X PBS, at 4°C O/N (1 oligo:3 Ab) . Storage: At 4°c in 0.01% NaN3 Read more
Authors: Rasel A. Al-Amin and Ulf Landegren
+1
+1
-1

Pages