Molecular Cloning od Recombinant DNA - (Oct/26/2010 )

My class conducted the experiment for Molecular Cloning od Recombinant DNA and I just have a few questions

1. This experiment uses a HindIII mixture of insert fragments of different lengths ( and a vector of pUC18 vector with is 2.7kb). but it is difficult to obtain recombinant plasmids with either of the two largest fragments clones, the 23.1 and 9.4 kb fragments. give two reasons for this difficulty.

2.Our results for bench 4 experiment in which vector was prepared by a double restriction digest ( EcoRI + BamHI) to generate different ends for zero background colonies. if you performed a parallel experiment using a different enzyme pair SmaI/HaeIII to prepare the vector, whuch was then ligated to fragments with SmaI/HaeIII ends. What results would be ontained when the transformation plates were examined?
( i think this is due too both enzymes being blunt ended...and since directional cloning with blunt wont work, but why wont they work with blunt ended enzyme? and how would be plate look due to this?)

3. In the plasmid purification section, the plasmid is purified from the other components of the cleared lysate by being bound to hydroxylated silica in the column disc, in the presence of a buffer that also contains salt. after the washing step, the plasmid is eluted in pure water. how is the elution in pure water achieved?

I would greatly appriciate answeres from any questions, thank you!

Hmmm - as with most forums, you will find that people here will be more willing to help if you at least have a go at answering the questions yourself.

generally speaking in this forum we are more likely to help you when there are technical issues, after you had try to solve it but doesn't work out.

I assume you are still doing your undergraduate.vIt will be good if you ask the questions in homework section.

Your question 1 sounds like a homework question: why you would ever need exactly "two reasons"? What if there is only one?
my brief answer: when you try to ligate a large fragment into vector, there are lots of technical stuff you should be aware: fragment vs size ratio, fragment purity etc...

In Your question 2, I don't understand what you trying to ask... also you are asking how the plate looks like... general rule: if there was disrupted lacZ gene (fragment cloned in)... you will not get blue colony in Amp plate if is a success clone.

For Your question 3: Do you know how exactly the DNA binding to silica column work? In fact, if you ask here: what is the minimum requirement of salt concentration in binding buffer... I think most of the veteran like Bob1 will more happy to discuss with you. Look into most of the silica-based kit's manual, and you will find the answer. If you still can't get the answer, ask again and I will tell you.



Just a gentle reminder, if you think Bob1 is trolling, see how he professionally reply all the other technical questions and most of the time his advices had helped many in their research work. There are definitely many professional molecular geeks here (yes, is true: post docs, professors, etc) whom had published many good quality journals.

lolcakes on Wed Oct 27 02:10:33 2010 said:

That reply's a bit harsh (especially to someone who has close to 1,600 posts here) and is not likely to motivate people here to get involved in answering your question...

Bob1 has accurately stated the policy of this forum with regards to questions that are (or appear to be) homework questions: we will not just answer the questions for you, but will guide you to the correct answers if you provide us with some of your thoughts on what the correct answers might be, and why you're unsure of your conclusions.