lookiecono.blogg.se

1. #Serial cloner construct digest only one end how to#
2. #Serial cloner construct digest only one end manual#
3. #Serial cloner construct digest only one end full#

Then select “pGGAselect” from the destination vector's dropdown menu. Add the destination vector by clicking on “Add Sequence”. In our example, we will select “BsmBI-v2” and use sequences that have no internal sites.Īdd the destination vector. If this is not possible, view our video on Golden Gate Domestication for strategies dealing with internal sites. Choose the restriction enzyme that has no internal sites in the DNA sequences to be used in the assembly. Enzymes available from NEB are shown in red.

#Serial cloner construct digest only one end full#

The tool will help design PCR primers to make amplicon inserts, check sequences for internal Type IIS restriction enzyme cut sites, and generate a report that shows the assembly graphically, and includes a full construct sequence file.īegin by selecting the Type IIS restriction enzyme you have chosen for your assembly design from the dropdown menu.

As an example, we will build an assembly from five sample inserts or modules using the pGGAselect destination plasmid supplied with our Golden Gate Assembly Kits.

#Serial cloner construct digest only one end how to#

In this video, we will demonstrate how to use the NEB Golden Gate Assembly Tool. Pool the positive mini-preps if you need a larger amount of plasmid for an experiment.Tutorial for NEB Golden Gate Assembly Tool. Screen plasmids by restriction digest to confirm correct size and lack of additional unexpected bands, then sequence positive clones if necessary.You might find that some plasmids give a very good yield and others less so, however the low yield plasmids are often correct. Elute plasmid in ~30 µl of water or elution buffer.You may need to vortex the culture tube thoroughly as I find the bacteria are often quite sticky. Some cultures may show disappointing levels of growth compared to others, but still go ahead with your mini-prep protocol and you should get a bacterial pellet. Use entire 3ml culture to do plasmid extraction.Pick a mixture of both, but in my experience the slower-growing colonies contain the correct construct.

Note that on your plate there may be a mixture of large, fast-growing colonies and smaller colonies that take longer to appear.

#Serial cloner construct digest only one end manual#

I checked the gateway manual and it said (see attached picture 2) that sometimes the CCDB gene gets mutated and becomes chlora-resistant. Not sure why? Shouldn't it not grow on chlora? Sequencing showed that the gene was indeed in the plasmid. I transformed one of the maxipreps I had on an amp plate and did the same with a chlora plate and they both grew colonies. Therefore, true positive clones would ONLY grow on ampicillin plates. Thus, this eliminates the positions from attr1 through attr2.removing the gene encoding chloramphenicol resistance and inserting your cDNA there. From my understanding, during the LR reaction.your entry clone containing the cDNA of interest gets integrated into the destination vector (pDEST15) which happens through a process that involves flanking the two attr sites on pDEST15. We are using gateway cloning for protein expression using pDEST15 as our destination vector. I am having a few doubts about gateway cloning. After the incubating stage, out of 4 plates, only two plates had colonies (but each plate only had like 2 visible colonies), and these two plates were the ones with 100 ul transformation. For each sample, one plate had 50 ul of transformation mix + 150 ul LB broth, and for the other plate I doubled the amount of transformation mix, which was 100 ul, and then added 100 LB broth. Specifically, in the transformation after adding outgrow medium, I diluted each sample I got with LB broth, meaning I had two plates per sample. Next, I carried out transformation to collect colonies to then do mini prep and restriction digest to validate. I got two samples so far, which again were exactly the same. The next step I did was BP reaction to create entry clones. At this stage, I had two PCR products, and they were exactly the same I had an extra in case I might mess up. I carried out PCR to generate attB-PCR product, which was treated with DpnI and purified. I had a plasmid, PHIP, with a specific concentration I got beforehand.