Nayeem991

You can isolate the plasmid in large volume (midi method, following the protocol mentioned in molecular cloning by maniatis et al). you will get higher amount of plasmid. Where do you get the single band in the gel (size of your undigested band)? do you get isolated band after digestion or it just become smear? If it is smear i am afraid the band you see is the band of chromosomal DNA because generally we see 3 bands for undigested plasmid.

Did you use X-gal and IPTG in your selection media. If you don't use IPTG your inserted gene(streptomycin resistance gene) wont be expressed because the plasmid contains tac promoter and also contains lacI which inhibits the promoter. So, check this step. On the other hand i would suggest you to select your transformed cell in double antibiotic(Ampicillin and streptomycin) as your plasmind contains Amp resistance.

First confirm the orientation of insertion. I think it will work if both the promoters are in same direction (as phage 434 said) and the gene will obviously express under the promoter of vector.  If they are in opposite direction i think it will only work if the plasmid is transformed into the host from where your gene is originally belongs to. Because that host contains the particular RNA pol which will recongnise the promoter and it will express the gene inserted in either direction. But i guess in this case it wont fulfill your purpose.

tihong10, on 21 July 2012 - 10:51 AM, said:

Actually the baseline reading is not a constant thing. whenever you perform an experiment you should take 2 sets of samples. One with the test material and another is without test material which works as control. Then you have to compare them to get an idea about the effect of your test material on expression of the particular gene. Suppose you have 4 mutants A, B, C and D (you don't know where the transposon has inserted)and you want to study the effect of increased salt concentration on these 4 mutants. For this experiment you have to take two sets of sample. Set 1 would be with normal salt concentration and set 2 would be with increased salt concentration. Then comparing the result of set 2 with set 1 you can identify which mutant is affected by increased salt concentration (expression level may increase of decrease). You may take multiple copies of both sets for better comparison of your result. Suppose expression level of B is increased due to increased salt concentration, then you can identify what actually mutant B is (Where the transposon has inserted). So, identification of insertion site is not important for initial screening. But the fact is, this kind of transposon is generally used to create a mutant library. In that case you  have to identify the insertion site first. Because to create a library you need to have mutant of every possible genes of that organism (except the genes responsible for the survival of that organism). If you read the paper (which i recommended) you would be able to understand how the mutant library is created.  Is it clear to you now? please let me know if there is any further query....

tihong10, on 19 July 2012 - 05:08 PM, said:

your understanding is correct. Actually commercial kit is available to assay beta galactosidase activity and it is expressed as "Miller Unit" after some mathmatical calculation. You can google it for better understanding.   The gene in which the transposon is inserted is identified by two round of semiarbitrary PCR followed by sequencing. The junction sequence is first identified and then the gene is identified using some bioinformatics tools. For better understanding you can read this paper.... http://www.pnas.org/...05/25/8736.full I hope it will help you for better understanding.About your second question I would like to say that, in every biological experiment control is an essential part. If you want to determine the effect of a particular thing on expression of your gene of interest, you need to use a control where you wont add that particular thing. Suppose you want to assay the expression level of a particular gene in response to increased salt concentration. In that case you  will fix the baseline of expression at normal salt concentration then you will compare that with increased one. Is it clear to you now?