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Team:Washington/General Cloning Strategy
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For our cloning strategies we chose to do restriction digestion not only because we are using the standard bio-brick, but also because we want to teach our students the most classic cloning methods. | For our cloning strategies we chose to do restriction digestion not only because we are using the standard bio-brick, but also because we want to teach our students the most classic cloning methods. | ||
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Instead of heat inactivating both of the digestions we have the insert PCR purified and the vector gel purified. This will significantly reduce the colonies of self-ligated undigested vectors and insert plasmid into the next step. The vector and insert are best ligated by the ratio of 1: 3 (make the vector 2 uL and the insert 6 uL). We transformed the ligation into the cells by the method of heat shock instead of electro competent transformation since it cost less and was easy to perform. For the heat shock transformation, 4 uL of ligation to 45 uL of the competent cell usually give a good result. During the recovery, in order to let the cell have enough time to produce the protein with the antibiotic, we found that 30 to 45 minutes give the highest efficiency. We also perform the recovery in a 1.5 micro-centrifuge tube instead of the PCR tube because the aeration is better and it helps the cell produce the protein more efficiently. | Instead of heat inactivating both of the digestions we have the insert PCR purified and the vector gel purified. This will significantly reduce the colonies of self-ligated undigested vectors and insert plasmid into the next step. The vector and insert are best ligated by the ratio of 1: 3 (make the vector 2 uL and the insert 6 uL). We transformed the ligation into the cells by the method of heat shock instead of electro competent transformation since it cost less and was easy to perform. For the heat shock transformation, 4 uL of ligation to 45 uL of the competent cell usually give a good result. During the recovery, in order to let the cell have enough time to produce the protein with the antibiotic, we found that 30 to 45 minutes give the highest efficiency. We also perform the recovery in a 1.5 micro-centrifuge tube instead of the PCR tube because the aeration is better and it helps the cell produce the protein more efficiently. | ||
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| + | After we have the parts cloned properly by confirming the sequencing result, we build the sensors with bio-bricks and test them on our red light; green light system. | ||
Revision as of 02:08, 25 September 2013
For our cloning strategies we chose to do restriction digestion not only because we are using the standard bio-brick, but also because we want to teach our students the most classic cloning methods.
We first grow the tabor cells overnight. These cells carry the plasmid we are targeting to clone into bio-brick vectors. We dilute DNA from the mini-prep to a low concentration, such as 1 ng/uL, so we can avoid having too many wrong sites during PCR. Therefore we don’t have to do DPN1 digestion. Then we mini-prep the DNA out and we design the primer PCR the targeted insert. We designed the primers by taking the first and last 18~35 bases and add the bio-brick prefix and suffix for each forward and reverse primers. We also found that in order to get it to work properly we need to add 5~6 bases for the reverse primer as an overhang.
Before we digested the PCR product we purified it with the PCR purification kit so that it enhanced the quality of the ligation product. Then we digest both the insert and the bio-brick vector with the restriction enzymes Xbal and Pst1 with buffer 3 and 4. We also did digestion using other enzyme Xbal and Pst1-HF with Cut-smart buffer, which we also resulted in similar efficiency.
Instead of heat inactivating both of the digestions we have the insert PCR purified and the vector gel purified. This will significantly reduce the colonies of self-ligated undigested vectors and insert plasmid into the next step. The vector and insert are best ligated by the ratio of 1: 3 (make the vector 2 uL and the insert 6 uL). We transformed the ligation into the cells by the method of heat shock instead of electro competent transformation since it cost less and was easy to perform. For the heat shock transformation, 4 uL of ligation to 45 uL of the competent cell usually give a good result. During the recovery, in order to let the cell have enough time to produce the protein with the antibiotic, we found that 30 to 45 minutes give the highest efficiency. We also perform the recovery in a 1.5 micro-centrifuge tube instead of the PCR tube because the aeration is better and it helps the cell produce the protein more efficiently.
After we have the parts cloned properly by confirming the sequencing result, we build the sensors with bio-bricks and test them on our red light; green light system.
