Why do I get smeared band after restriction?

12 December 2018 1 4K Report

I restricted digested 1microgram of my PCR product with two enzymes NcoI and XhoI. I incubated one vial overnight and another for 3 hours. The vial which was incubated overnight has got a crisp band but the one which was incubated for 3 hours got a hazy smear.. What could be the possible reasons?

Paul Rutland

possibly the enzyme is still bound to the dna in the shorter digestion causing different lengths of dna and the bound protein causing the dna to run at different speeds. In the longer digestion perhaps the enzymes have cut and fallen off leaving a cleaner product

Badges
Science method

More Soundarya Priya's questions See All

How do I remove supernatant completely from cell lysate?

I am using E.coli BL21 Plyss for a recombinant protein production. The cells debris becomes too sticky after lysis. I spin down the pellet at 13000 g (that is the maximum available here). The...

07 August 2019 3,528 7 View

Are there any standard protein-ligand titration reactions that I can use to calibrate my ITC equipment?

The acid base titration standards are working fine. I just want to know if there are any protein-ligand standards. Thanks in advance

04 May 2019 2,094 3 View

Can PCR cleanup kits be used for gel elution of restricted plasmid DNA?

I use PCR cleanup kit from Thermoscientific. The extraction of restricted plasmid from gel failed twice but the extraction of insert which was run parallely gave me DNA with good concentration and...

01 February 2019 1,982 18 View

Can a HiPrep 16/60 Sephacryl S-100 column be connected to peristaltic pumps?

Can I connect the column to a peristaltic pump operated at a low flow rate and leave the other end open to elute the contaminants out of the column? Or will it disrupt the column packing??

01 February 2019 9,756 6 View

Can I run a restriction digestion just by increasing enzyme volume and not reaction volume?

I need a huge concentration of restricted product. So can i just digest them in a 50 uL reaction with high volume of enzyme (say 5-7uL). This is just to run the digested product in a single gel...

01 February 2019 2,068 13 View

What are the points I need to take care when designing primers to amplify GOI which will be used for cloning?

The crucial points that need to be considered to avoid failures in cloning.

31 December 2018 9,980 2 View

What are the desired Self complementarity and Self 3' complementarity values?

What is the acceptable range when checking the primers using Primer-BLAST

31 December 2018 6,994 4 View

How to unclog FPLC column?

The HiPrep 16/60 Sephacryl S-100 column that we use is clogged with some dark coloured particles at the very top. I tried cleaning up the column with 0.5M NaOH followed by distilled water. This...

31 December 2018 5,444 9 View

Is it necessary to add oligos front and back of the restriction sites?

Should I add oligos based on the chart flanking both the ends of the restriction site or adding oligos to the end of DNA is enough?

31 December 2018 2,665 6 View

Does absorbance at 230nm influence cloning?

I have purified my PCR product with PCR purification kit by thermo scientific. When analysing the sample in nanodrop after purification I get a very high absorbance at 230 nm. Will this interfere...

11 December 2018 9,371 8 View

Protein-aptamer gel electrophoresis help, please??

Hi, I have problems with running gel electrophoresis. I have tried agarose gel electrophoresis and native PAGE. I have two proteins, which have molecular weights of ~30kDa and ~180kDa and two...

03 March 2021 4,275 4 View

Enhanced Yellow Fluorescent Protein (Aequorea victoria) DNA sequence?

I am on the lookout for the Enhanced Yellow Fluorescent Protein (Aequorea victoria) DNA sequence. Does anyone know where I can find it? Thank you in advance

03 March 2021 3,568 1 View

Can linear DNA be used as template for site-directed mutagenesis?

Is it possible to induce site-directed substitution mutation by quick-change method on linear dsDNA? or it has to be cloned in some vector? If yes, should it be treated with the Dpn1 enzyme...

03 March 2021 401 4 View

How do I increase the yield of my PCR product?

Hello, We would like to increase the yield of our PCR product. We are running a series of PCR reactions that is targeting ~1.1kb sequence. We begin each reaction with ~400pg of template DNA...

02 March 2021 4,029 3 View

Gel electrophoresis result for total RNA samples, 1.5 Agarose gel was used, How to improve the integrity of RNA?

Gel electrophoresis, RNA degradation, RNA extraction from fresh tissue

02 March 2021 5,433 5 View

New Journal, "Integrative Psychological and Behavioral Science" -- do you not know, and have you not seen, this done before?

Question to you and THEM, the New Journal, "Integrative Psychological and Behavioral Science" -- do you not know, and have you not seen, this done before? There appears to be a core problem for...

02 March 2021 3,024 2 View

Research on Supply Chain Management in Indian context?

Hi, I am planning to apply for the PhD degree in the Supply Chain Mgt. with specific area of "Cold Storage warehouses" during Pandemics and wars. Where lock downs and shut downs are frequent....

02 March 2021 285 2 View

PACYC PCR not working after several attempts. Would anyone have any suggestions?

So, I have been trying to run a pACYC PCR which will be used later on for a Gibson Assembly. However the PCR is not working. I have already tried gradient PCR and changing extension time; however...

02 March 2021 1,146 2 View

How to regulate SSCP temperature in simple PAGE?

I'd like to perform single-strand conformation polymorphism (SSCP) in my thesis, however I cannot control the temperature of the vertical PAGE since we are using the conventional tanks. Is there a...

02 March 2021 9,157 1 View

Does anyone have a recommendation for yeast reporter gene plasmid/protocol?

Hi, could anyone recommend a plasmid and/or protocol for reporter gene assay in S. cerevisiae? I want to assess the effect of growth conditions on a transcription factor, so I want to clone it`s...

01 March 2021 210 1 View