Hot answers tagged cloning
9
This is a great question as I just made my own "homebrew" chemically competent cells.
There are a vast variety of E. coli strains that are commonly used for cloning. They may be transformed chemically by heat shock method, or electrically by electroporation (a brief summary may be found here). These can be made in the lab manually, or purchased commercially ...
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I have heard that Epi300 electrocompetent cells (1) from Epicentre are very efficient: > 1 x 1010 cfu/µg of pUC19. Dan Gibson used them in his paper for the synthesis of the mitochondrial genome (2). We were also thinking of using them for our assemblies, but they are pretty expensive.
1. TransforMax™ EPI300™ Electrocompetent E. coli
2. Chemical synthesis ...
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Yes, you must fragment the genome in order to insert it into a vector for cloning; you can't "insert" the whole 5 Mbp genome of E. coli into a vector. It's difficult to transform cells with huge plasmids, 2-20 kbp is an optimal range. In any case, if you want a clone of the whole genome, wait 30 minutes and the cell will happily oblige you.
Most ...
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For the impatient, I'm going to say that probably if you only have a regular home freezer, its unlikely that you will be able to do this. If you have access to a -80C or liquid nitrogen storage its possible, but also less likely.
Cloning an animal typically starts with the transplantation of a nucleus into an egg cell whose nucleus has been removed. So ...
5
Did you identify structural domains in the protein using Pfam or some other domain detecting peptide search?
It's pretty useful to cut your constructs at the boundaries of structurally independent domins in the protein if you know where they are. Hydrophobicity plots might lead you to cut right in the middle of a protein fold, only exascerbating inclusion ...
5
I have lots of experience with the TOPO TA kit which is very similar to the kit your using. Here's a link to the TOPO Directional cloning product page which shows a schematic of the enzymatic ligation.
From your comments, you said you have doubly-digested the PCR product. This is likely your point of failure unless you are absolutely confident that your ...
5
I think the best way is option #2:
Suppose that your gene of interest is AAAAAAAAAAAAAAAAAAAAAAAGGGGGGGGGGGGGGGGGGGGGGGG
and you want to insert EcoRI restriction site GAATCC
Then your Fwd primer will be GAATCC AAAAAAAAAAAAAAAA
and your Rev primer will be GGATTC CCCCCCCCCCCCCCCC
But in general, it shouldn't matter which option you choose, as long as you ...
5
Try dissolving at 50C. In the Qiagen gel extraction kit, it says to dissolve at 50C or until completely dissolved.
The size of the gel is important too. Make sure it is 1.5% or less, and the size of the gel needs to be less than 400 mg.
Also, pay attention to the first step. If the liquid is not the same color as the color of the first QG buffer then ...
5
When I optimized gel extraction in my hands, two factors turned out to be critical in maximizing gel extraction yield:
The pH of the eluting solution. I used to elute the final product with DEPC-treated water initially, but the yield would vary a lot as the pH of the water changed. Using buffer EB is better, since it maintains a stable pH 8.
Most ...
2
In my own personal lab experience, I got some unexpected results using Phusion polymerase. However, I have not seen deletions, only insertions (ranging from single bases to 3 tandem copies of a primer sequence). This is not an answer to your deletion question, but it does suggest unusual things might happen.
I have seen deletions with T4 DNA polymerase ...
1
Disclaimer: I haven't ever done a Gibson assembly, but here is my theoretical understanding of how to design your primers. You need four 40mers each consisting of 20 bp segments derived from the vector and the insert and corresponding to the junctions that you are trying to create. In the diagram below the dotted lines represent the junctions between the two ...
1
The neurological basis of behavior is still unclear, and the genetic basis of behavior is even less clear. What can be said is that genetics may predispose you to certain behaviors, but what the predisposing factors are, and how much they determine your behavior (and how much is due to environmental factors), is unclear. And this varies for different ...
1
You can look up Gibson Assembly or Circular Polymerase Extension cloning (CPEC). For both of these the website for J5 has some good protocols. Here is the one for CPEC: http://j5.jbei.org/j5manual/pages/80.html
For CPEC you can look at the 2011 Quan paper: http://www.ncbi.nlm.nih.gov/pubmed/21293463
Hopefully that helps.
1
On the question "would this possible?", my answer is yes.
It is not unimaginable that organs could be grown without MHC proteins, although, as you mentioned, there's no guarantee that essential functions of the organ wouldn't be affected as well.
This is not science-fiction at all. The question deserves a good answer by someone who works in the field!
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