BIOLOVER

Creating sgRNA library using ssDNA bridging Efficient Cloning with NEBuilder HiFi and ssDNA Oligos Save time and resources in your lab by using NEBuilder HiFi DNA Assembly with single-stranded oligos (ssDNA) . This guide explains how to bypass expensive dsDNA synthesis services for your cloning projects. Two Powerful ssDNA Strategies Strategy A. Multiple Oligo Assembly: Overlap several short ssDNAs (60nt each) to form a long, functional DNA fragment. Strategy B. ssDNA Bridging: Use a single ssDNA oligo to bridge gaps between linearized vector ends, ideal for sgRNA-Cas9 constructs. Step-by-Step Protocol 1. Oligo Design & Annealing Design each oligo with a 30nt overlap to its neighbor. 5' phosphorylation is NOT required. Prepare in 1X NEBuffer 2: Step 1: Mix 100uM oligos (Final concentration ~0.2uM) ...

The Mystery of Gel Bands: Ghost Bands Without DNA? (Solving Loading Dye & Protein Artifacts) The Mystery of Gel Bands: Ghost Bands Without DNA? (Solving Loading Dye & Protein Artifacts) Have you ever scratched your head while checking your gel electrophoresis results? You look at a sample where you expect no DNA, yet a mysterious band that looks exactly like DNA appears, leaving you confused. "Is it contamination?" "Did I make the gel wrong?" Countless worries might cross your mind. But don't panic. This phenomenon is more common than you think, and the culprit is often closer than you expect. It is highly likely caused by the unfortunate meeting of your habitual Loading Dye and residual proteins in your sample. Based on discussions with our experts, today we will clearly explain why these Artifacts (Ghost Bands) occur and how you can achieve clean r...

DNA Circularization Guide: Succeeding with HiFi Assembly for RCA Templates DNA Circularization Guide: Succeeding with HiFi Assembly for RCA Templates Hello fellow researchers! Are you struggling at the bench today trying to circularize a PCR-amplified DNA fragment ? Creating a clean circular DNA template without a vector, especially for Rolling Circle Amplification (RCA) , can be trickier than it sounds. Today, let's discuss a specific scenario: You have a ~700 nt gene fragment, Golden Gate Assembly failed you, and now you are looking into HiFi DNA Assembly as a solution. 💡 Key Question: "Did Golden Gate fail because of the 4bp sticky end? Can HiFi Assembly circularize a single fragment without a vector?" 1. Golden Gate Failure: Is the 'Short Sticky End' Really to Blame? Many researchers suspect that the 4bp o...

Luna qPCR Optimization: Complete Primer Design Guidelines (Including Tm, Amplicon, GC Content) Luna qPCR Optimization: Complete Primer Design Guidelines (Including Tm, Amplicon, GC Content) Hello, all researchers in the bio lab!🧪 qPCR (Quantitative Polymerase Chain Reaction) , often called the 'master key' to quantitative gene expression analysis—how often do you use it? The success of qPCR depends on many factors, like reagent choice and equipment conditions, but the most critical is 'Primer Design' . It's like the architectural blueprint! Are you currently using Luna qPCR products? Today, I will provide detailed and specific Primer Design Guidelines optimized for the Luna qPCR product line. I'll make sure to fill it with core information so it can rank high in search results. I'm confident that reading this article to the end will noticeably improve your qPCR efficiency! ...

NEBExpress T4 Lysozyme vs Chicken Lysozyme: Ultimate Strategies for Gram-Positive Cell Lysis & DNA Extraction Purity NEBExpress T4 Lysozyme vs Chicken Lysozyme: Ultimate Strategies for Gram-Positive Cell Lysis & DNA Extraction Purity 🔬 Introduction: The Success of Your Experiment Hinges on Cell Lysis! If you've ever performed DNA extraction or protein purification in the lab, you know how crucial the initial cell lysis step is. This is especially true when dealing with Gram-positive bacteria , which are notorious for their robust cell walls! Today, I'm going to provide a detailed comparison of two powerful cell lysis protocols offered on the NEB website: one using Chicken Lysozyme and another utilizing NEBExpress T4 Lysozyme . Furthermore, I'll reveal advanced tips to enhance the purity of your extracted samples. Get ready to discover key insights that will maximize your experimental effici...

FFPE 시료 DNA 손상, 완벽하게 복구하는 NEBNext® FFPE DNA Repair v2 모듈 가이드 안녕하세요, 바이오 연구자 여러분!🔬 오늘은 까다로운 FFPE(Formalin-Fixed Paraffin-Embedded) 시료를 다루는 분들을 위해 아주 유용한 정보를 가져왔습니다. FFPE는 보존성이 뛰어나지만, DNA가 심하게 손상되는 문제가 있어 차세대 염기서열 분석(NGS)과 같은 정밀한 실험에 사용하기 어렵다는 단점이 있습니다. 특히 DNA 탈아미노화(deamination) 와 같은 손상은 염기 서열을 왜곡하여 잘못된 분석 결과를 초래할 수 있습니다. 하지만 걱정하지 마세요! NEBNext® FFPE DNA Repair v2 Module 이 손상된 DNA를 효과적으로 복구하여 여러분의 실험 성공률을 크게 높여줄 수 있습니다. 이 글을 통해 FFPE 시료의 DNA 손상 원리와 복구 메커니즘, 그리고 NEBNext의 솔루션까지 자세히 알아보겠습니다. 왜 FFPE 시료의 DNA는 손상될까? FFPE(포르말린 고정 파라핀 포매) 과정은 조직을 영구적으로 보존하는 데 탁월합니다. 하지만 이 과정에서 사용되는 포르말린(formalin) 은 DNA에 치명적인 손상을 입힙니다. 포르말린은 DNA 가닥을 서로 교차 결합(cross-linking)시키고, 염기쌍을 변형시키며, 탈아미노화(deamination) , 산화(oxidation) , 닉(nicks) , 그리고 AP(abasic) sites 와 같은 다양한 손상을 유발합니다. 특히 탈아미노화 는 시토신(cytosine)이 우라실(uracil)로 변환되는 현상으로, PCR이나 NGS 과정에서 우라실이 티민(thymine)으로 오인식되어 염기 서열에 오류를 일으킵니다. 이러한 손상들은 유전체 분석의 정확성을 크게 ...

Complete DNA Damage Repair in FFPE Samples: NEBNext® FFPE DNA Repair v2 Module Guide Is Damaged FFPE DNA Derailing Your NGS? Unlock High-Quality Results with NEBNext® FFPE DNA Repair v2 Module! Hello, fellow bioscience researchers! 🔬 Today, I've brought some incredibly useful information for those of you working with challenging FFPE (Formalin-Fixed Paraffin-Embedded) samples. While FFPE samples offer excellent preservation, they often suffer from severe DNA damage, making them difficult to use for precise experiments like Next-Generation Sequencing (NGS). In particular, DNA damage such as deamination can distort base sequences, leading to erroneous analysis results. But don't worry! The NEBNext® FFPE DNA Repair v2 Module can effectively repair damaged DNA, significantly increasing your experimental success rate. In this article, we'll delve into the causes of DNA damage in FFPE samples, the repair m...