Double Digestion with EcoRI and XhoI?

More Thomson Patrick Joseph's questions See All

Reason (s) for retraction of articles?

Recently, through social media i came across several issues of article retraction. Where, blots were even duplicated, FACS results were duplicated, blots were manipulated and so on.... What are...

02 March 2018 7,715 4 View

Microarray Data Analysis?

Hello, Which software can be used (free) for the analysis of micro-array data other than GESA and GenePattern ? Best Regards

01 February 2018 4,073 3 View

HEK293 and HEK293T cell lines?

What is the difference between HEK293 and HEK293T cells? HEK293T is also written as 293T cells? Thanks Regards

11 December 2017 3,268 1 View

Complex I activity determination of mitochondria?

Hello, What method can be used for the determination of complex I activity of mitochondria (cultured cells/fresh tissue)? Thanks Best Regards

11 December 2017 7,703 4 View

Mitochondrial DNA and whole mitochondrial genome sequencing?

For the whole mitochondrial genome sequencing, how i can achieve the good quality of mt DNA from animal tissue? Any suggestion Regards

11 December 2017 2,564 2 View

Principle Component Analysis (PCA) Analysis?

Which software can be used for PCA analysis? where i can draw the graph and interpretate it? Any guideline how PCA analysis can be done using SPSS... Regards

10 November 2017 6,501 20 View

Difference between the SCI (Science Citation Index) Journal and SCIE (Science Citation Index Expanded)?

What is the basic difference between between the SCI (Science Citation Index) Journal and SCIE (Science Citation Index Expanded)? Does it matters in case of quality of journal? Thanks Regards

10 November 2017 1,460 9 View

Criteria for the distribution of Impact Factor between author(s)?

What is the criteria of IF distribution between the authors? It varies from institution to institution or there is any fix rule? If the IF of journal is 5.0 how it will be distributed between...

10 November 2017 2,500 4 View

Paper should be written before/after thesis?

Hello, What is advisable for a PhD student to write thesis first or publish first? or both can be done simultaneously? Best Regards

10 November 2017 9,329 4 View

HEK-293T can be used as normal cell line?

HEK-293T cell line can be used as normal cell line if i am investigating the anticancer potential of a compound against a panel of cancerous cell line (considering HEK-293T as normal among the...

10 November 2017 4,416 3 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

Why might my GFP tag be dissociating from my plasmid after transfection?

I have a set of stably transfected cell lines all transfected with plasmids containing GFP tags on the C terminus. During a western blot using anti-GFP antibody, one of my plasmids has dissociated...

01 March 2021 9,310 4 View

Expression cloning by using cDNA,do I need to modify the cDNA first?

I am going to have a expression cloning of mammalian gene by using shuttle plasmid to transforming the E.coli However I don't know I should only inserting the Coding sequence ,or I can...

28 February 2021 5,440 3 View

Problem with shRNA transfection. Why are only two plates getting contaminated instead of all?

I transfected my LNCaP-WT cells with 3 shRNA plus their NTC two weeks ago and split two puromycin selected cell plates on Friday last week(Feb 26). I checked for GFP in the cells, and they all...

28 February 2021 4,949 3 View

Anyone that can help with details in phosphopeptides enrichment?

Anyone know the amount of protein before enzymatic digestion for phosphopeptides enrichment of serume sample in Ti-IMAC? Has anyone ever use Ti-IMAC(the material is from Chinese company named...

28 February 2021 7,310 3 View

PUC19 as a transfer plasmid for lentiviral delivery?

Hello, Is it possible to use pUC19 as a transfer vector to be packed in using the second generation viral particles packaging system( pMD2.G; psPAX2 plasmids)? As far as I understand it there is...

28 February 2021 4,868 2 View

Did anyone ever try using pLVX-TetOne Plasmid with a copGFP as a selection marker instead of Puromycin?

When using a lentiviral vector for inducible expression of genes using the Tet-On system. In the literature, I saw a widely used of pLVX-TetOne-Puro Plasmid, is there a reason why people prefer to...

25 February 2021 1,011 3 View

Why do the PCR bands from the cDNA templates (extraction from the cell after transfection) show different size from the band of the plasmid template?

After transfection with the plasmid ( linearized ) and subcloning of the cell lines, RNA was extracted from the cell and then reverse transcripted to cDNA. When PCR reactions were run to verify...

25 February 2021 5,712 3 View

How do I transfect a large plasmid into PC12 cells with nerve growth factor (NGF) receptor?

I have a large 16 kb plasmid, which I need to transfect into PC12 cells. Lipofectamin 2000 didn't work and with GFP alone the transfection rate is very low. I also tried the neon invitrogen...

25 February 2021 6,635 5 View

Problem with ligating 2.8 kb insert into 8.5 kb plasmid?

I am trying to clone 2 copies of the same mammalian gene into the pSF-CMV-Ub-Puro Ascl (contains HindIII, KpnI and NheI cut sites) plasmid. This is what the final product is supposed to look like...

24 February 2021 6,310 3 View

Haifa Juma

this link can help you

https://www.neb.com/tools-and-resources/interactive-tools/double-digest-finder

Thomson Patrick Joseph

hello...thank you....

Tomáš Hluska

The set-up of your reaction would be helpful (which buffer, how much of everything etc.)

Does your band have the size of plasmid+insert? If so, one of your enzymes is not working. For that you should always do single-digest controls. Simply do the same reaction, but add only one of the enzymes. And you can do it only in 5 ul to save material. In that case you will immediately know what went wrong.