Anyone familiar with restriction double digestion of p35S-FAST Plasmid?

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What are the primers to get whole length of the sequence?

Dear all Please if someone can guide me.. I have cloned a gene of 1500 bp pairs in FAST-Plasmid. I used reverse primers to sequence the gene. The company sent me 900bp length.. I want to sequence...

11 December 2015 8,729 1 View

Restriction double digestion of p35S-FAST Plasmid..?

Hello every one I want to double digest p35S-FAST Plasmid with Kpn1 and BamH1 1X tango buffer.. The "Double Digest Calculator-Thermo Scientific" recommends the following condition. 1X Tango...

10 November 2015 4,454 0 View

Which (forward or reverse, or both) primers should I use for cDNA synthesis from gene specific primers?

I have extracted RNA from plant immature fruit. I want to synthesize its cDNA by gene specific primers. Please guide me on which (forward or reverse or both) primers are used in such cases.

03 April 2014 9,362 6 View

Can you help me achieve adequate amplification of required band?

I am using 0.2mM dNTPs,40pm primers,0.6microlitre Taq,2 microlitre MgCl2(2.5mM) in a total 20 microlitre reaction volume for PCR. I have kept the temperature the same so far. I need a band size of...

02 March 2014 1,460 13 View

Would the process of extracting RNA from plants without liquid nitrogen be successful?

02 March 2014 4,430 9 View

Would RNA extraction from plants be successful if i do not use Liquid nitrogen?

Thanks in advance

02 March 2014 3,995 1 View

How should my cDNA band look like on gel?

I want to synthesize cDNA from plant RNA. Please tell me that what should the cDNA band look like on gel, so that I can guess that cDNA has successfully synthesized. Please show me some gel...

01 February 2014 887 1 View

Can anyone help with RNA storage?

I stored plant RNA samples at -20 degree centigrade for 5 days and then I shifted to -80 degrees. Can I use this RNA for cDNA synthesis?

01 February 2014 6,004 7 View

Can anyone help with optimizing PCR conditions for my gene of interest?

I made different dilutions of control cDNA and in each case after running its PCR got the band of 496bp .The Gel pic is as below. But when I ran PCR for my gene of interest with same dilutions of...

01 February 2014 652 3 View

Can anyone help with cDNA agarose gel?

I have synthesised cDNA from plant RNA .The gel picture is given below. Please if anyone could tell me that whether the cDNA has synthesised or not? If synthesised, can I use this for Reverse...

01 February 2014 3,377 13 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

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

Best plasmid could be used as a standard curve for AAV titration?

I have been trying to have a good AAV titration for my production and I have tried different plasmids to set up as a standard curve but each one behaves differently. i am trying to use ITR for...

23 February 2021 5,167 3 View