Name: streamlined purification of plasmid dna from prokaryotic cultures
Uploaded: 2011-01-05T17:00:00.000+00:00
Duration: 6 min 53 s
Description: Watch this Scientific Journal Video about Streamlined Purification of Plasmid DNA From Prokaryotic Cultures at JoVE.com

1. Lysate 통관 <ol><li> E. 그로 37 밤새 적절한 항생제로 Luria의 수프에 깊은 우물을 접시에 원하는 플라스미드 DNA (문화 / 잘 ​​1 ML) · 잡고 C로 변형 대장균 문화. </li><li> 10 분 5,000 XG에 문화 접시에있는 펠렛 E. 대장균, 다음 가만히 따르다 뜨는. </li><li> 100 μL Resuspension 버퍼 (50 MM 트리스 - HCL, pH를 8.0, 10 MM EDTA (에틸렌 다이아 민 테트라 초산), 100 μg / ML RNase A)의 각 펠렛을 Resuspend. </li><li> 100 μL / 잘 용해 버퍼 (200 MM NaOH, 1 % SDS) 추가하고 2 분 동안 접시 통을 사용하여 발라줍니다. </li><li> 100 자 / μL 중화 버퍼 (3.0 M의 칼륨 아세테이트, 산도 5.5) 추가하고 2 분 흔들. </li><li> 설명 판을 lysate로 세포 lysate을 전송합니다. </li><li> 350 μL 수집 플레이트 및 진공 매니폴드의 하단에 자리 위에 플레이스 DNA 바인딩 플레이트. (그림 1 참조) 진공 매니폴드를 조립. </li><li> 진공 장치 위에 플레이스 lysate의 명확 플레이트. </li><li> HG (0.34 바) 인치 10 진공을 적용하고 DNA 바인딩 플레이트에 여과물를 수집합니다. HG 인치 12보다 큰 진공 청소기를 사용하지 마십시오. (0.41 바). </li><li> 진공 매니폴드를 분해. 장치의 아래쪽에 넣어 두 ML 폐기물 수집 플레이트. </li></ol> 2. DNA 바인딩 <ol><li> (그림 2 참조) 다기관의 맨 ​​위에 DNA 바인딩 플레이트를 이동합니다. </li><li> 300 μL / 잘 바인딩 버퍼 (6 M 구아니딘 - HCL)에 추가하고 혼합하고 아래로 피펫. </li><li> 진공을 적용 [~ 느린 진공에 대한 HG 인치 5 (0.17 바)]와 여과물을 삭제. DNA는 이제 막에 바인딩됩니다. </li><li> 와시 버퍼 (80 % 에탄올)의 / 음 400 μL로 씻으십시오. </li><li> 진공을 적용 후 여과액 폐기하십시오. 2 ML 수집 플레이트를 사용하거나 낭비 직접 필터링하는 경우 불필요 폐기하십시오. </li><li> 한번 세차를 반복합니다. </li><li> 건조 흡수 타월에 필터 플레이트의 하단에 얼룩. </li><li> 잔류 알코올의 제거를 위해 5-10분 위해 한번 더 진공 적용됩니다. </li><li> 에탄올 방울의 제거를 위해 바닥 얼룩. </li></ol> 3. DNA의 용출 <ol><li> 70 자 / μL 용출 버퍼 (10 MM 트리스 - HCL, pH를 8.0, 1 ㎜ EDTA (에틸렌 다이아 민 테트라 초산)) 추가합니다. 높은 DNA 농도가 원하는 경우, 볼륨은 50 μL / 음에 줄일 수 있지만, 평균 총 수확량이 낮은 것입니다. </li><li> 일분 실온에서 접시를 품어. </li><li> 정화의 DNA는 진공 또는 원심 분리에 의해 eluted 수 있습니다. </li><li> 진공 방식 : 진공 매니폴드에 플레이스 깨끗한 수집 판 (DNase, RNase 무료). 모든 용출 버퍼 DNA 바인딩 플레이트 통과 때까지 진공 매니폴드 위에 플레이스 필터 플레이트는 1 분 HG (0.5 바) 인치 15 진공을 적용합니다. 정화의 DNA를 (그림 3 참조)를 수집합니다. </li><li> 원심 분리 방법 : 5 분 동안 1,000 XG에 깨끗한 수집 판과 원심 분리기 위에 플레이스 정화 필터 플레이트. </li></ol> 4. 양적 및 질적 분석 <ol><li> OD 280분의 260 측정, DNA를 정화의 농도를 계산합니다. </li><li> 정화 pDNA의 아가로 오스 겔의 분석을 수행합니다. </li></ol> 5. 대표 결과 E. 대장균이 자료를 시작 파편 감지할 수있을 정도의 감소를 보여 기존의 원심 분리와 팔 AcroPrep 사전 lysate의 명확 필터 플레이트를 사용하여 명확히 lysate. (그림 4). 세 농도에서 플라스미드 DNA는 TE 버퍼로 아군과 AcroPrep 사전 lysate의 명확 필터 플레이트 통과되었다. 플라스미드 DNA 농도의 비교, 사전 및 사후 여과, 쇼는 세 가지 농도 100 % 복구 (그림 5) ~. 명확히 E.에서 DNA의 복구를 입증하기 위해 대장균 lysate, 플라스미드 DNA가 원유 lysate 치솟았어 후 여과하여 명확히했다. 동일한 플라스미드 DNA가 세포 lysate의 다음 여과에 추가되었습니다. 전기는 각 샘플 (그림 6)에서 aliquots에서 수행되었다. 각 샘플에서 유사한 강도의 하나, 명확 밴드는 여과 기반 설명의 단계에서 작은 경우에는 모든 DNA 손실을 보여줍니다. E.의 문화 pCAT 플라스미드 DNA를 포함하는 대장균은 E.에서 정화되었습니다 대장균은 팔의 350 μL 1 ML의 DNA 바인딩 필터 플레이트뿐만 아니라, 350 μL DNA 바인딩 접시 두 가지 다른 브랜드를 사용 lysates. 제조 업체에서 권장하는 프로토콜에 명시된 바와 같이 모든 purifications이 수행되었다. 가장 높은 DNA 농도와 총 생산량은 팔의 1 ML AcroPrep 사전 DNA 정화 필터 플레이트 (그림 7과 표 2)에서 볼 수 있습니다. 경쟁 W 또한 높은 DNA 농도를 (155 NG / μL)를 보여줍 있지만, 전체 DNA의 수율이 (4.7 μg / 음) 낮은 복구 볼륨으로 인해 가장 낮은이며, ~ 30 μL. 경쟁 M 108에서 DNA의 가장 낮은 농도를 준 5.6 μg / 음의 총 수율과 μL / NG. 한천세 가지 DNA 바인딩 접시에서 pCAT DNA를 정화의 오세 겔 분석 그림 8에 표시됩니다. 각 수집된 샘플 볼륨이 전기하기 전에 복구 볼륨의 차이에 대한 정확한 65 μL로 조정되었습니다. 모든 샘플은 플라스미드 DNA를 supercoiled 보여주지만, 필터 플레이트의 각 DNA의 수율이 달라집니다. 그림 9는 준비 DNA가 supercoiled 양식에 모두 있으며, 유사한 농도를 가지고 보여줍니다. <img src="/files/ftp_upload/2407/2407fig1.jpg" alt="그림 1" /> 그림 1. Lysate 자세한 내용을 진공 매니폴드의 조립. <img src="/files/ftp_upload/2407/2407fig2.jpg" alt="그림 2" /> 그림 2. DNA 바인딩에 대한 진공 매니폴드의 조립. <img src="/files/ftp_upload/2407/2407fig3.jpg" alt="그림 3" /> 그림 3. 진공 용출을위한 진공 매니폴드의 조립. <img src="/files/ftp_upload/2407/2407fig4.jpg" alt="그림 4" /> 그림 4. 여과는 샘플 허가를 단순화합니다. 진공 여과 (350 μL AcroPrep 사전 lysate의 명확 필터 플레이트) 또는 전통적인 원심 분리에 의해 명확히 원유 lysate 300 μL의 세중의 샘플. OD 320 설명 전후에 측정. <img src="/files/ftp_upload/2407/2407fig5.jpg" alt="그림 5" /> 그림 5. AcroPrep 사전 lysate의 명확 필터 플레이트를 통해 통과 후 pDNA의 전체 복구. 300 μL TE 버퍼는 25, 50 및 100 μg / ML에서 pCAT를 타서. DNA 농도 및 복구, N = 2 전, 여과 후 OD 260에서 계산. <img src="/files/ftp_upload/2407/2407fig6.jpg" alt="그림 6" /> 그림 6. 설명 후 pCAT DNA의 복구. 이전이나 이후에 설명 lysate 치솟았어 pUC19 동등한 금액은 다음 TE 버퍼 10에 1을 희석. 1.2 % 아가로 오스 겔 2 μL / 레인로드됩니다. <img src="/files/ftp_upload/2407/2407fig7.jpg" alt="그림 7" /> 그림 7. 팔 Acroprep 사전 DNA 경쟁 플레이트보다 구속력이 필터 플레이트 잘마다 높은 플라스미드 DNA를 얻을 수 있습니다. 1 ML (팔와 경쟁 업체의 M) 1.5 ML (경쟁 W)의 야간 문화 표시 DNA 정화 플레이트를 사용하여 pCAT 플라스미드 DNA의 수율 (OD 260) DH5α. 강판 제조 업체의 권장 프로토콜을 사용하여 정제. 오차 막대는 표준 오류 (N&gt; 6) 나타냅니다. <img src="/files/ftp_upload/2407/2407fig8.jpg" alt="그림 8" /> 그림 8. pCAT의 DNA의 아가로 오스 겔 분석 표시된 DNA 바인딩 접시에 정화. 정화 pDNA의 1.2 % 아가로 오스 겔 전기 영동. 세 가지 purifications에서 풀링된 샘플은 eluate는 정화, 희석 1시 10분 후 65 μL 조정. 레인 당 2 μL 일만 했어. <img src="/files/ftp_upload/2407/2407fig9.jpg" alt="그림 9" /> 그림 9. 품질 및 원심 분리기 또는 진공 방법을 정화 pDNA 최종 eluate 수집 아가로 오스 겔 전기 영동에 사용하는 경우와 비슷한 순수한 DNA의 복구. AcroPrep 사전 필터 플레이트 제조 업체에서 권장하는 프로토콜은 pDNA 정화에 대한 미행했다. 용리 2 분 HG (0.5 바) 인치 15 오분이나 진공 1,000 XG에서 원심 분리기에 의해 수행되었다.

이 비디오 문서의 저자는 악기와 비디오 및 텍스트에 등장하는 시약을 생산 팔 생명 과학의 직원입니다.

우리는 몇 가지 장점을 내실수 AcroPrep 사전 필터 플레이트의 손쉬운, 간소화된 프로토콜을 설명합니다. 수동 세미 자동, 또는 완전 자동 AcroPrep 사전 형식으로 처리하면이 방법은 최대 플라스미드 DNA의 수율을 제공합니다. lysate 통관 접시에 통합 prefilter도 샘플, 입자의를 포함 높은 수준의 일관성있는 여과를 제공합니다. 보다 빠르고 균일 최소한의 개최 - 최대 볼륨으로 접시에 걸쳐 여과 속도에 판 결과 잘 도형 및 아울렛 팁 기하학은 교차 오염의 우려없이 수신기 플레이트에 샘플을 직접 흐름을 제공합니다. 중요한 AcroPrep 사전 필터 플레이트의 DNA 결합 판의 높은 바인딩 용량 막은 높은 품질의 플라스미드 DNA의 최대 회복에 최적입니다.

<table><tbody><tr><td>AcroPrep Advance Filter plate</td><td>Pall Corporation</td><td>PN 8175 (1 mL) or PN 8075 (350 &amp;#181;l)</td><td>3 &amp;#x03BC;m glass fiber 0.2 &amp;#x03BC;m Supor&amp;#174; membrane for clarification</td></tr><tr><td>AcroPrep Advance Filter Plate</td><td>Pall Corporation</td><td>PN 8132</td><td>for DNA Binding</td></tr><tr><td>Vacuum manifold</td><td>Pall Corporation</td><td>PN 5017</td><td></td></tr><tr><td>Vacuum pressure pump</td><td>Pall Corporation</td><td>PN 13157</td><td></td></tr></tbody></table>

streamlined purification of plasmid dna from prokaryotic cultures

우리는 prokaryotic 문화에서 시작하고 고순도 DNA로 끝나는, 96 플라스미드 준비를위한 AcroPrep 사전 필터 플레이트의 전체 과정을 설명합니다. 세균성 lysate 통관 및 DNA의 정화를위한 멀티 잘 여과에 따라이 방법은 플라스미드 준비를위한 능률적인 프로세스를 만듭니다. 실리카 기반의 미디어를 포함하는 필터 플레이트는 쉽게 진공 여과 또는 플라스미드 DNA가 감지할 수있을 정도의 수량을 항복하기 위해 원심 분리기에 의해 처리할 수 있습니다. 정량 분석은 정화 플라스미드 DNA가 1.97의 평균 OD 280분의 260 비율로 양질의 지속 결정. 전반적으로, 플라스미드 수율은 시퀀싱 및 복제와 같은 다운 스트림 응용 프로그램에 대한 더 순수한 DNA를 제공합니다. AcroPrep 사전 필터 번호판을 사용하는 이것은 능률적인 방법은, 수동 세미 자동 또는 완전 자동으로 처리 수 있습니다.

Watch this Scientific Journal Video about Streamlined Purification of Plasmid DNA From Prokaryotic Cultures at JoVE.com

Streamlined Purification of Plasmid DNA From Prokaryotic Cultures

이 프로토콜은 효율적으로 병렬에서 설명 및 플라스미드 DNA의 정화를위한 비용 효율적인 대안입니다<em&gt; E. 대장균</em&gt; 문화. AcroPrep 사전 프로세스가 높은 바인딩 용량 DNA 바인딩 필터를 접시에 정화 다음 최적의 lysate의 명확 필터 플레이트와 함께 시작합니다.

Prokaryotic 문화 플라스미드 DNA의 유선형 정화

We describe the complete process of AcroPrep Advance Filter Plates for 96 plasmid preparations, starting from prokaryotic culture and ending with high ...

streamlined-purification-of-plasmid-dna-from-prokaryotic-cultures

Research

JoVE Journal

Biology

19.9K 조회수.  Pall Life Sciences.  中文 

비디오: Streamlined Purification of Plasmid DNA From Prokaryotic Cultures

A G-quadruplex DNA-affinity Approach for Purification of Enzymatically Active G4 Resolvase1

Higher-order nucleic acid structures called G-quadruplexes (G4s, G4 structures) can form in guanine-rich regions of both DNA and RNA and are highly thermally stable. There are &#62;375,000 putative G4-forming sequences in the human genome, and they are enriched in promoter regions, untranslated regions (UTRs), and within the telomeric repeat. Due to the potential for these structures to affect cellular processes, such as replication and transcription, the cell has evolved enzymes to manage them. One such enzyme is G4 Resolvase 1 (G4R1), which was biochemically co-characterized by our laboratory and Nagamine et al. and found to bind extremely tightly to both G4-DNA and G4-RNA (Kd in the low-pM range). G4R1 is the source of the majority of G4-resolving activity in HeLa cell lysates and has since been implicated to play a role in telomere metabolism, lymph development, gene transcription, hematopoiesis, and immune surveillance. The ability to efficiently express and purify catalytically active G4R1 is of importance for laboratories interested in gaining further insight into the kinetic interaction of G4 structures and G4-resolving enzymes. Here, we describe a detailed method for the purification of recombinant G4R1 (rG4R1). The described procedure incorporates the traditional affinity-based purification of a C-terminal histidine-tagged enzyme expressed in human codon-optimized bacteria with the utilization of the ability of rG4R1 to bind and unwind G4-DNA to purify highly active enzyme in an ATP-dependent elution step. The protocol also includes a quality-control step where the enzymatic activity of rG4R1 is measured by examining the ability of the purified enzyme to unwind G4-DNA. A method is also described that allows for the quantification of purified rG4R1. Alternative adaptations of this protocol are discussed.

G4 Resolvase1 binds to G-quadruplex (G4) structures with the tightest reported affinity for a G4-binding protein and represents the majority of the G4-DNA unwinding activity in HeLa cells. We describe a novel protocol that harnesses the affinity and ATP-dependent unwinding activity of G4-Resolvase1 to specifically purify catalytically active recombinant G4R1.

효소 활성 G4 Resolvase1의 정화를위한 G-quadruplex DNA 화성 접근

Higher-order nucleic acid structures called G-quadruplexes (G4s, G4 structures) can form in guanine-rich regions of both DNA and RNA and are highly ...

연구

생화학

Purifying Plasmid DNA from Bacterial Colonies Using the Qiagen Miniprep Kit

Plasmid DNA purification from E. coli is a core technique for molecular cloning. Small scale purification (miniprep) from less than 5 ml of bacterial culture is a quick way for clone verification or DNA isolation, followed by further enzymatic reactions (polymerase chain reaction and restriction enzyme digestion). Here, we video-recorded the general procedures of miniprep through the QIAGEN's QIAprep 8 Miniprep Kit, aiming to introducing this highly efficient technique to the general beginners for molecular biology techniques. The whole procedure is based on alkaline lysis of E. coli cells followed by adsorption of DNA onto silica in the presence of high salt. It consists of three steps: 1) preparation and clearing of a bacterial lysate, 2) adsorption of DNA onto the QIAprep membrane, 3) washing and elution of plasmid DNA. All steps are performed without the use of phenol, chloroform, CsCl, ethidium bromide, and without alcohol precipitation. It usually takes less than 2 hours to finish the entire procedure.

Qiagen Miniprep 키트를 사용하여 세균의 플라스미드 식민지에서 DNA를 정화

Plasmid DNA purification from E. coli is a core technique for molecular cloning. Small scale purification (miniprep) from less than 5 ml of bacterial ...

생물학

A Purification and In Vitro Activity Assay for a (p)ppGpp Synthetase from Clostridium difficile

Kinase and pyrophosphokinase enzymes transfer the gamma phosphate or the beta-gamma pyrophosphate moiety from nucleotide triphosphate precursors to substrates to create phosphorylated products. The use of &#947;-32-P labeled NTP precursors allows simultaneous monitoring of substrate utilization and product formation by radiography. Thin layer chromatography (TLC) on cellulose plates allows rapid separation and sensitive quantification of substrate and product. We present a method for utilizing the thin-layer chromatography to assay the pyrophosphokinase activity of a purified (p)ppGpp synthetase. This method has previously been used to characterize the activity of cyclic nucleotide and dinucleotide synthetases and is broadly suitable for characterizing the activity of any enzyme that hydrolyzes a nucleotide triphosphate bond or transfers a terminal phosphate from a phosphate donor to another molecule.

A Purification and In Vitro Activity Assay for a pppGpp Synthetase from Clostridium difficile

Here, we describe a method for purifying histidine-tagged pyrophosphokinase enzymes and utilizing thin layer chromatography of radiolabelled substrates and products to assay for the enzymatic activity in vitro. The enzyme activity assay is broadly applicable to any kinase, nucleotide cyclase, or phosphor-transfer reaction whose mechanism includes nucleotide triphosphate hydrolysis.

정화 및 체 외에서 활동 분석 결과 (p) ppGpp Clostridium 남과 어울리지 않 는에서 합성에 대 한

Kinase and pyrophosphokinase enzymes transfer the gamma phosphate or the beta-gamma pyrophosphate moiety from nucleotide triphosphate precursors to ...

면역학 및 감염병학

High-throughput Purification of Affinity-tagged Recombinant Proteins

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further biochemical analyses to obtain detailed insights into structure/function relationships. Advances in oligonucleotide- and gene synthesis technology make large-scale mutagenesis strategies increasingly feasible, including the substitution of target residues by all 19 other amino acids. Gain- or loss-of-function phenotypes then allow systematic conclusions to be drawn, such as the contribution of particular residues to catalytic activity, protein stability and/or protein-protein interaction specificity.
In order to attribute the different phenotypes to the nature of the mutation - rather than to fluctuating experimental conditions - it is vital to purify and analyse the proteins in a controlled and reproducible manner. High-throughput strategies and the automation of manual protocols on robotic liquid-handling platforms have created opportunities to perform such complex molecular biological procedures with little human intervention and minimal error rates1-5.
Here, we present a general method for the purification of His-tagged recombinant proteins in a high-throughput manner. In a recent study, we applied this method to a detailed structure-function investigation of TFIIB, a component of the basal transcription machinery. TFIIB is indispensable for promoter-directed transcription in vitro and is essential for the recruitment of RNA polymerase into a preinitiation complex6-8. TFIIB contains a flexible linker domain that penetrates the active site cleft of RNA polymerase9-11. This linker domain confers two biochemically quantifiable activities on TFIIB, namely (i) the stimulation of the catalytic activity during the 'abortive' stage of transcript initiation, and (ii) an additional contribution to the specific recruitment of RNA polymerase into the preinitiation complex4,5,12 . We exploited the high-throughput purification method to generate single, double and triple substitution and deletions mutations within the TFIIB linker and to subsequently analyse them in functional assays for their stimulation effect on the catalytic activity of RNA polymerase4. Altogether, we generated, purified and analysed 381 mutants - a task which would have been time-consuming and laborious to perform manually. We produced and assayed the proteins in multiplicates which allowed us to appreciate any experimental variations and gave us a clear idea of the reproducibility of our results.
This method serves as a generic protocol for the purification of His-tagged proteins and has been successfully used to purify other recombinant proteins. It is currently optimised for the purification of 24 proteins but can be adapted to purify up to 96 proteins.

We describe a method for the affinity-tagged purification of recombinant proteins using liquid-handling robotics. This method is generally applicable to the small-scale purification of soluble His-tagged proteins in a high-throughput format.

선호도 태그 재조합 단백질의 높은 처리량 정화

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further ...

Engineering Antiviral Agents via Surface Plasmon Resonance

Surface plasmon resonance (SPR) is used to measure hemagglutinin (HA) binding to domain-swapped Cyanovirin-N (CV-N) dimer and to monitor interactions between mannosylated peptides and CV-N's high-affinity binding site. Virus envelope spikes gp120, HA, and Ebola glycoprotein (GP) 1,2 have been reported to bind both high- and low-affinity binding sites on dimeric CVN2. Dimannosylated HA peptide is also bound at the two low-affinity binding sites to an engineered molecule of CVN2, which is bearing a high-affinity site for the respective ligand and mutated to replace a stabilizing disulfide bond in the carbohydrate-binding pocket, thus confirming multivalent binding. HA binding is shown to one high-affinity binding site of pseudo-antibody CVN2 at a dissociation constant (KD) of 275 nM that further neutralizes human immunodeficiency virus type 1 (HIV-1) through oligomerization. Correlating the number of disulfide bridges in domain-swapped CVN2, which are decreased from 4 to 2 by substituting cystines into polar residue pairs of glutamic acid and arginine, results in reduced binding affinity to HA. Among the strongest interactions, Ebola GP1,2 is bound by CVN2 with two high-affinity binding sites in the lower nanomolar range using the envelope glycan without a transmembrane domain. In the present study, binding of the multispecific monomeric CV-N to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) glycoprotein is measured at KD = 18.6 &#181;M as compared with nanomolar KD to those other virus spikes, and via its receptor-binding domain in the mid-&#181;-molar range.

Engineering Antiviral Agents via Surface Plasmon Resonance

The present protocol describes new tools for SPR binding assays to examine CV-N binding to HA, S glycoprotein, related hybrid-type glycans, and high-mannose oligosaccharides. SPR is used to determine the KD for binding either dimeric or monomeric CV-N to these glycans.

표면 플라즈몬 공명을 통한 항바이러스제 엔지니어링

Surface plasmon resonance (SPR) is used to measure hemagglutinin (HA) binding to domain-swapped Cyanovirin-N (CV-N) dimer and to monitor interactions ...

생체공학

Plasmid Stability Analysis with Open-Source Droplet Microfluidics

Plasmids play a vital role in synthetic biology by enabling the introduction and expression of foreign genes in various organisms, thereby facilitating the construction of biological circuits and pathways within and between cell populations. For many applications, maintaining functional plasmids without antibiotic selection is critical. This study introduces an open-hardware-based microfluidic workflow for analyzing plasmid retention by culturing single cells in gel microdroplets and quantifying microcolonies using fluorescence microscopy. This approach allows for the parallel analysis of numerous droplets and microcolonies, providing greater statistical power compared to traditional plate counting and enabling the integration of the assay into other droplet microfluidic workflows. By using plasmids expressing fluorescent proteins alongside a non-specific fluorescent DNA stain, single colonies can be identified and differentiated based on plasmid loss or fluorescent marker expression. Notably, this advanced workflow, implemented with open-source hardware, offers precise flow control and temperature management of both the sample and the microfluidic chip. These features enhance the workflow&#39;s ease of use, robustness, and accessibility. While the study focuses on Escherichia coli as the experimental model, the method&#39;s true potential lies in its versatility. It can be adapted for various studies requiring fluorescence signal quantification from plasmids or stains, as well as for other applications. The adoption of open-source hardware broadens the potential for conducting high-throughput bioanalyses using accessible technology in diverse research settings.

An accessible, open-source microfluidic workflow is presented for the parallelized analysis of plasmid retention in bacteria. By employing fluorescence microscopy to quantify plasmid presence in single-cell microcolonies within gel microdroplets, this method provides a precise, accessible, and scalable alternative to traditional plate counting.

Open-Source Droplet Microfluidics를 이용한 플라스미드 안정성 분석

Plasmids play a vital role in synthetic biology by enabling the introduction and expression of foreign genes in various organisms, thereby facilitating ...

Use of In Vivo Assembly for High-efficiency Plasmid Construction

In vivo assembly (IVA) is a molecular cloning method that uses intrinsic enzymes present in bacteria that promote intermolecular recombination of DNA fragments to assemble plasmids. This method functions by transforming DNA fragments with regions of 15-50 bp of homology into commonly used laboratory Escherichia coli strains and the bacteria use the RecA-independent repair pathway to assemble the DNA fragments into a plasmid. This method is more rapid and cost-effective than many molecular cloning methods that rely on in vitro assembly of plasmids prior to transformation into E. coli strains. This is because in vitro methods require the purchase of specialized enzymes and the performance of sequential enzymatic reactions that require incubations. However, unlike in vitro methods, IVA has not been experimentally shown to assemble linear plasmids. Here we share the IVA protocol used by our laboratory to rapidly assemble plasmids and subclone DNA fragments between plasmids with different origins of replication and antibiotic resistance markers.

In vivo assembly is a ligation-independent cloning method that relies on intrinsic DNA repair enzymes in bacteria to assemble DNA fragments by homologous recombination. This protocol is both time and cost-effective, as few reagents are required, and cloning efficiency can be as high as 99 %.

고효율 플라스미드 구조를 위한 In Vivo Assembly 사용

In vivo assembly (IVA) is a molecular cloning method that uses intrinsic enzymes present in bacteria that promote intermolecular recombination of DNA ...

Plasmid-derived DNA Strand Displacement Gates for Implementing Chemical Reaction Networks

DNA nanotechnology requires large amounts of highly pure DNA as an engineering material. Plasmid DNA could meet this need since it is replicated with high fidelity, is readily amplified through bacterial culture and can be stored indefinitely in the form of bacterial glycerol stocks. However, the double-stranded nature of plasmid DNA has so far hindered its efficient use for construction of DNA nanostructures or devices that typically contain single-stranded or branched domains. In recent work, it was found that nicked double stranded DNA (ndsDNA) strand displacement gates could be sourced from plasmid DNA. The following is a protocol that details how these ndsDNA gates can be efficiently encoded in plasmids and can be derived from the plasmids through a small number of enzymatic processing steps. Also given is a protocol for testing ndsDNA gates using fluorescence kinetics measurements. NdsDNA gates can be used to implement arbitrary chemical reaction networks (CRNs) and thus provide a pathway towards the use of the CRN formalism as a prescriptive molecular programming language. To demonstrate this technology, a multi-step reaction cascade with catalytic kinetics is constructed. Further it is shown that plasmid-derived components perform better than identical components assembled from synthetic DNA.

This protocol describes a method for deriving DNA strand displacement gates from plasmids and testing them using fluorescence kinetics measurements. Gates can be modularly composed into multi-component systems to approximate the behavior of formal chemical reaction networks (CRN), demonstrating a new use for CRNs as a molecular programming language.

플라스미드 유래 구현 화학 반응 네트워크 용 DNA 가닥 변위 게이츠

DNA nanotechnology requires large amounts of highly pure DNA as an engineering material. Plasmid DNA could meet this need since it is replicated with high ...

Genome-wide Purification of Extrachromosomal Circular DNA from Eukaryotic Cells

Extrachromosomal circular DNAs (eccDNAs) are common genetic elements in Saccharomyces cerevisiae and are reported in other eukaryotes as well. EccDNAs contribute to genetic variation among somatic cells in multicellular organisms and to evolution of unicellular eukaryotes. Sensitive methods for detecting eccDNA are needed to clarify how these elements affect genome stability and how environmental and biological factors&#160;induce their formation in eukaryotic cells. This video presents a sensitive eccDNA-purification method called Circle-Seq. The method encompasses column purification of circular DNA, removal of remaining linear chromosomal DNA, rolling-circle amplification of eccDNA, deep sequencing, and mapping. Extensive exonuclease treatment&#160;was&#160;required for sufficient linear chromosomal DNA degradation. The rolling-circle amplification step by &#966;29 polymerase enriched for circular DNA over linear DNA. Validation of the Circle-Seq method on three S. cerevisiae CEN.PK populations of 1010 cells detected hundreds of eccDNA profiles in sizes larger than 1 kilobase. Repeated findings of ASP3-1, COS111, CUP1, RSC30,&#160;HXT6, HXT7 genes on circular DNA in both S288c and CEN.PK suggests that DNA circularization is conserved between strains at these loci. In sum, the Circle-Seq method has broad applicability for genome-scale screening for eccDNA in eukaryotes as well as for detecting specific eccDNA types.

This paper presents a sensitive method called Circle-Seq for purifying extrachromosomal circular DNA (eccDNA). The method encompasses column purification, removal of remaining linear chromosomal DNA, rolling-circle amplification and high-throughput sequencing. Circle-Seq is applicable to genome-scale screening of eukaryotic eccDNA and studying genome instability and copy-number variation.

진핵 세포에서 염색체 외 원형 DNA의 게놈 차원의 정화

Extrachromosomal circular DNAs (eccDNAs) are common genetic elements in Saccharomyces cerevisiae and are reported in other eukaryotes as well. EccDNAs ...

CAPRRESI: Chimera Assembly by Plasmid Recovery and Restriction Enzyme Site Insertion

Here, we present chimera assembly by plasmid recovery and restriction enzyme site insertion (CAPRRESI). CAPRRESI benefits from many strengths of the original plasmid recovery method and introduces restriction enzyme digestion to ease DNA ligation reactions (required for chimera assembly). For this protocol, users clone wildtype genes into the same plasmid (pUC18 or pUC19). After the in silico selection of amino acid sequence regions where chimeras should be assembled, users obtain all the synonym DNA sequences that encode them. Ad hoc Perl scripts enable users to determine all synonym DNA sequences. After this step, another Perl script searches for restriction enzyme sites on all synonym DNA sequences. This in silico analysis is also performed using the ampicillin resistance gene (ampR) found on pUC18/19 plasmids. Users design oligonucleotides inside synonym regions to disrupt wildtype and ampR genes by PCR. After obtaining and purifying complementary DNA fragments, restriction enzyme digestion is accomplished. Chimera assembly is achieved by ligating appropriate complementary DNA fragments. pUC18/19 vectors are selected for CAPRRESI because they offer technical advantages, such as small size (2,686 base pairs), high copy number, advantageous sequencing reaction features, and commercial availability. The usage of restriction enzymes for chimera assembly eliminates the need for DNA polymerases yielding blunt-ended products. CAPRRESI is a fast and low-cost method for fusing protein-coding genes.

Here, we present chimera assembly by plasmid recovery and restriction enzyme site insertion (CAPRRESI), a protocol based on the insertion of restriction enzyme sites into synonym DNA sequences and functional plasmid recovery. This protocol is a fast and low-cost method for fusing protein-coding genes.

카스트레 : 플라스미드 회수 및 제한 효소 부위 삽입에 의한 키메라 조립

Here, we present chimera assembly by plasmid recovery and restriction enzyme site insertion (CAPRRESI). CAPRRESI benefits from many strengths of the ...