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At beginning of the week was decided the best way to insert a prefix-RFP-suffix fragment to thepBBR1MCS-5 plasmid, which is a plasmid with broad host-range and is capable to conjugate to R.
So we want to eliminate the most of the restriction sites and obviously we need to eliminate the prefix and suffix sites (italic).
In this week we did plasmid isolations again, but rather using mini-preps we used a Roche kit, this in order to obtain plasmid clean enough to further digestion. We spent the rest of the week doing the ligation between the pBBR1MCS-5 and the prefix-RFP-suffix fragment and doing the further transformation. In this week the ligation and transformation of the digested clean PCR(prefix-RFP-suffix) and pBBR1MCS-5 was repeated. In order to see if the new plasmid properly contains the prefix and suffix a digestion with EcoR1 was made, this was successful.
Electrophoresis gel showing the insert (prefix-RFP-suffix) and plasmid (pBBR1MCS-5) with their correct sizes. On this week negative results about the conjugation were obtain, attempts to fix and improve the alternative conjugation protocol were made, but they were unsuccessful.


Competent S17 cells were made and transformation of the new plasmid (pBBR1MCS-5) into them was done.
We realized that when we amplify the RFP from the pSB1T3 we designed wrong our primers, because we also wanted to get the sequence previous the prefix and next the suffix where verification primers bind, but we did not get the sequence next de suffix, so a new primer was re-designed. 7 Comparison of cloned efficiency between non-optimized protocol (A) and optimized protocol (B).
No restriction site or recombination site needed, insert fragments generated by either PCR or restriction enzyme digestion can be used. Once the plasmid isolations were done it was proceeded to do a double digestion using apaI and sacI(see week: june 13 to 18). But we failed to obtain any colony with our insert probably because of the dirty PCR, so we did the PCR(prefix-RFP-suffix) again. BL21 was done in order to digest with ApaI both, the transformation and the digestion were successful. It consists to watch the percentage of bacteria without any selection that loose the plasmid.


In just 15 minutes at room temperature, any PCR fragment can be cloned into your linearized vector at will.
After done, the transformation of the pBBR1MCS-5 using the previous competent cell was done and further plasmid isolations were also done. After a simple clean up step, a PCR-generated DNA fragment or other purified DNA fragment can be joined to a vector with overlapping ends (Fig.1). The PCR products can be produced by either Taq DNA polymerase or other high fidelity DNA polymerase. Seven DNA fragments of different lengths were cloned into same vector respectively by Fast-Fusion Cloning Kit.



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