Och

please has anyone tracked the accummulation of aluminium in fruits using morin staining? I have two sets of fruit samples- one set treated with aluminum, and the other is not treated. normally the treated fruit shld show fluorescence when stained with morin since Al forms a green fluorescence complex with morin, while the non-treated should show little or no fluorescence. But when I observe the fruits under a fluorescence microscope after staining with morin at exciter 400-450 and emission 500-550, both the treated and non-treated fruits showed green fluorescence all over the fruits. Am confused on what actually the problem is. i really need assistance. Thanks.

the vector from which the insert is derived is encoded with amp resistance. Yes, the insert was cut out from an existing vector. Per further treatment, do you mean gel purification?

Thanks CharonY. When I want to ligate the unpurified vector, should I purify the insert or should I also leave it unpurified? That is, should I ligate the vector and the insert without purifying any of them? Also, do you think I can get the gene cloned onto the vector if I purify the vector(spectinomycin resistance) and do not purify the insert (ampicillin resistance)? Thanks.

@ CharonY. If for example I have 20ng vector DNA with 8000bp and 10ng insert with1600bp; so i will say 20*8000: 10*1600? how will I know the volumes of each to add to the ligation mix? So if I transform the digest(do you mean without ligation), should I see colonies? per the first step, do you mean transforming the plasmid after extraction from bacterial liquid culture? what is the reason for re-ligating the vector without agarose purification (with and without adding insert) and transforming it? Thanks a lot. I really appreciate your response.

@ CharonY. Thanks a lot for your invaluable reply. I will do some controls. I did not test the transformation. What I meant was that I tested the competent cells to make sure that they're okay. What do you mean by the concentration ratio of the of the insert to vector? I've not been able to figure out how to calculate the conc. ratio of the insert to vector. I will appreciate if you can help me with that. Please what do you mean by 're-ligate the vector without agarose purification '? Do you mean that I should ligate the vector after plasmid extraction and enzyme digestion? Thanks.

Hi all, Please I'm having some very serious problems on my project. What I am trying to do is to: 1. extract an EGFP gene from pSAT1-EGFP-C1 (4553 bp) and clone it onto the binary vector, pPZP-RCS2-ocs-bar-R1 (8637 bp)and then transform it with competent cells. 2. Later on, I will use it to clone some genes from tomato plants by silencing usin VIGS. But the problem I am having now is in the first step. I havn't been able to clone the EGFP gene onto the binary vector. The protocol I have been using is: For the vector (a) plasmid extraction of vector (plasmid conc. is usually high) (b) enzyme digestion of vector with Asc1 enzyme (I get one band after cutting) © agarose gel purification of vector (d) measurement of vector DNA concentration using nanodrop (sometimes I get low conc.) For the insert (a) plasmid extraction of insert (plasmid conc. is usually high) (b) enzyme digestion of insert also with Asc1 enzyme (I get 2 bands after cutting, then I use the lower band, with size of like 1600bp, for gel purification) © agarose gel purification of insert (d) measurement of insert DNA conc. (also I get very low conc. of DNA) Then I go ahead with ligation of the vector and insert by adding 4ul of insert to 1ul or 2ul of vector; 1ul of T4 DNA ligase; 1ul of T4 DNA ligase buffer; and add up with ddH2O in a 10ul reaction and incubate @ 16oC overnight. After that, I then go ahead with transformation using competent cells that were ordered and tested. I grow the ligation mix on spectinomycin selective media since the vector has spectinomycin resistance for 16 hours. But at the end, I will either: not see any growth; or see some king of very tiny colonies that won't grow on LB media. Please, I really need the help of this forum to know what the prob is. I have been stuck here for several months now. I will really appreciate all kinds of opinions on this.

Hi distinguished members, I'm pleased to sign in as a new member. I believe I can get some help from this forum regarding my research problem and also assist others in theirs. Our contributions will go a long way to help us all to achieve great things. No contribution to help solve a problem is too small or useless. Thanks guys