Can BpiI FD enzyme be used for Golden Gate assembly?

09 September 2018 0 3K Report

I use T4 ligase buffer for Golden Gate because it doesn't contain PEG. However, I don't know its compatibility with BpiI. I was wondering if anyone has experience with this?

Badges
Science method

More Ani Ben's questions See All

Why does pSMPUW have trouble sequencing after being midiprepped?

Hello. Recently I've been assembling plasmids that use pSMPUW as the backbone (kanamycin resistant). I've noticed that the minipreps for these plasmids usually sequence fine (I've been using stbl3...

03 April 2019 6,522 2 View

EF1alpha promoter compatible in DH5a cells?

I have a vector containing the EF1a promoter. I was wondering if DH5a cells will accept this vector upon transformation? I know some bacteria are incompatible with certain gene segments.

02 March 2018 2,251 8 View

Is there a more efficient assembly protocol for golden gate?

Hello. So for a current project I'm doing, I'm assembling several fragments (6-7) using golden gate assembly. I've combined the ligation and digestion in to one reaction, which takes ~5 hours in...

08 September 2017 1,691 2 View

Can we perform fragment assembly without primers?

Say you have three fragments that have overlaps with each other. During PCR, I'm assuming that since these fragments could physically overlap with each other they will anneal. My question is will...

07 August 2015 2,037 4 View

Buffer to disrupt platelet alpha granules for PF4 quantification?

Hello! I want to quantify by ELISA the secreted (from platelets poor plasma) and the non-secreted (from platelet lysate) PF4 before and after TRAP stimulation. I will use the ELISA from R&D...

03 March 2021 1,499 2 View

Buffer Exchange using column packed with sephadex G-25 effect on protein concentration?

Hello, If i am doing a buffer exchange for an antibody of 1mg/mL, does the elution lose protein in the process of buffer exchange? For example, if i flow through 500 uL of 1mg/mL sample, and...

03 March 2021 6,299 3 View

How optimize the Size Exclusion Chromatography?

Hello! I'll do a size exclusion chromatography, but I only have an open column, and I'll perform the peptide extraction from yeast, using buffer lysis (sodium phosphate 50 mM/NaCl 30 mM/DNAse and...

01 March 2021 2,215 2 View

Why are my bands weird in this Agarose gel?

Can someone please give me some possible things that could go wrong? Here is my recipe: 0.5g Agarose 50 mL of TAE 1x 1 uL ethyl bromide. Gel was run at 100V for 1 hour. The buffer used is also TAE.

01 March 2021 9,952 3 View

What do I do with DLS peaks that are from the buffer?

I'm looking at the aggregation of my protein sample using DLS. Unfortunately, my buffer (20mM HEPES) also results in a set of peaks. These are at approximately 1 and 1000 d.nm. The lowest peak...

01 March 2021 9,015 2 View

What is the most suitable resuspension buffer for TCA/acetone precipitated protein?

I need to extract protein from fermented carbon sources for Bradford assay. Most researchers experiencing insolubility of pellet in resuspension buffer. Please assist me to select most suitable...

26 February 2021 7,129 3 View

What urea buffer can i use for biotagged protein extraction from streptavidin beads?

Hi all, I have been doing research on biotagged LDB1 proteins in Mel cells. I purify the proteins using M280 streptavidin beads. The yield is very low so I've been trying to optimize it by...

24 February 2021 5,749 3 View

Cloudy/high background western blot - can anyone help me?

Hi everyone! I try to detect VDR protein (~48 kDa) using Western Blot. After SDS-PAGE using 10% MP TGX Stain-Free Gel from BioRad I transferred the protein onto an metOH activated PVDF membrane...

23 February 2021 6,266 3 View

How to improve the buffer capacity of sea water?

Hello everyone I would like to improve the buffer capacity of seawater (pH ~8) in order to dissolve as much CO2 as possible without dropping the pH below 7.5 (a range between 7 - 7.5 is...

17 February 2021 2,797 2 View

How can a reference point be fixed to an attachment point in Abaqus?

I am currently trying to copy an assembly feature, that is present in an old version of the model I am using, onto a new model. The feature is a reference point that is fixed to an attachment...

16 February 2021 8,872 2 View