The largest database of trusted experimental protocols

4 protocols using bacteriophage lambda dna

1

Biochemical Reagents for DNA Analysis

Check if the same lab product or an alternative is used in the 5 most similar protocols
All metal chlorides, formamide, and CAPS buffer were purchased from Sigma-Aldrich. Tris base was purchased from J. T. Baker and glacial acetic acid was from Mallinckrodt Chemicals. Ethylenediaminetetraacetic acid (EDTA)-disodium salt was obtained from EMD Chemicals, Inc. The 2-Log DNA ladder, also called 1 kb Plus DNA ladder, 1 kb Extend DNA ladder, bacteriophage lambda DNA and M13mp18 DNAs were purchased from New England Biolabs. E. coli chromosomal DNA (D2001) was obtained from Sigma-Aldrich. Agarose was from Gold Biotechnology.
+ Open protocol
+ Expand
2

Fluorescent Biosensor-Based Real-Time Helicase Assay

Check if the same lab product or an alternative is used in the 5 most similar protocols
Real-time helicase assays were performed using a fluorescent biosensor for ssDNA based on the Plasmodium falciparum SSB protein (fSSB; Chisty et al., 2018 (link)). Reactions were performed in RecBCD buffer (25 mM Tris-HCl pH 7.5, 10 mM NaCl, 6 mM MgCl2, 0.1 mg/ml BSA) supplemented with 10 pM RecBCD, 1 uM (in ntds; ~10 pM molecules) bacteriophage lambda DNA (New England Biolabs), 25 nM fSSB (tetramer) and gp5.9 at the stated concentration. After a 10 min pre-incubation, reactions were initiated by the addition of 2 mM ATP. Fluorescence intensity was monitored using a Cary Eclipse Fluorescence Spectrophotometer (excitation wavelength 430 nm, emission wavelength 475 nm, excitation and emission slit widths of 10 nm and 5 nm, respectively). Assays were performed in triplicate, and the initial rates reported are the mean and standard error for the three repeats. To obtain IC50 values for inhibition of RecBCD by gp5.9, data describing the initial unwinding rate as a function of log10[gp5.9] were fit to the sigmoidal dose–response equation of GraphPad Prism. rate=rmin+(rmaxrmin)/(1+10(logIC50log[gp5.9]))
rmin and rmax are the minimum and maximum DNA unwinding rates, respectively. Values for the unwinding rates were normalised to 100% for a zero gp5.9 control, and so for the purposes of the fits the values for rmax and rmin were constrained to 100 and 0 respectively.
+ Open protocol
+ Expand
3

Biotin-Labeling of Lambda DNA

Check if the same lab product or an alternative is used in the 5 most similar protocols
Bacteriophage lambda DNA from New England Biolabs was functionalized with biotin at both 5’ recessive ends following the standard protocol.26 ,27 (link) YOYO-1 and YO-PRO-1 were obtained from the Invitrogen and stored at −20°C before use. All experiments were done in a buffer of 0.01M HEPES, pH 7.4 (Acros Organics) including NaCl (Sigma-Aldrich) at the concentration specified when used. All water used was from a Barnstead E-Pure ultrapure water purification system with a resistivity of 18 MΩ cm−1.
+ Open protocol
+ Expand
4

Synthesizing DNA Fragments Using PCR

Check if the same lab product or an alternative is used in the 5 most similar protocols
We generated DNA of lengths 217 nm (639 bp), 398 nm (1170 bp), and 1023 nm (3008 bp) using PCR (36) . Specifically, we used bacteriophage lambda DNA (New England Biolabs N3011; Ipswich, MA) as a template, custom oligonucleotide primers (Integrated DNA Technologies; Coralville, IA), and an LA Taq DNA polymerase (TaKaRa Bio RR004; Kusatsu, Japan). We verified that products had amplified correctly using gel electophoresis and then extracted the DNA using a commercial kit (Qiagen QIAquick Gel Extraction Kit, 28704; Hilden, Germany). Finally, we measured the concentration and purity using a spectrophotometer (ThermoFisher NanoDrop Lite; Waltham, MA). Samples with A260/A280 ratios of less than 1.7 were discarded.
We purchased protamine from salmon (Sigma-Aldrich P4005; Saint Louis, MO), diluted it in deionized water, and stored 30 M aliquots at -20 C.
+ Open protocol
+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required

Sign up now

Revolutionizing how scientists
search and build protocols!