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Mutagenesis series: Ligation in Higher Volume

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  Introduced  The previous attempt to amplify a linear strand was unsuccessful.  There was 5 uL of DNA used as this amount was used previously when the ligation was successful, so a light band was expected to be seen on the gel. The assumption is that because the ligation was completed under certain conditions that this amplification process will need to be modified to fit those specific conditions. In this attempt, the Overlap process was used to remake the linear strand.  There were 6 tubes used to make enough of the sample to use for transformation.  Procedure   Using the 2-way assembly of the right+middle, the addition of the left fragment was introduced to the solution that consisted of a 5X Q5 master mix, primers 1 and 6, and PCR water.  This process went through Overlap PCR and the Final Fusion steps of the Overlap PCR protocol.  The conditions of the thermocycler were set to the prescribed temperatures and durations that are detailed in the Overlap PCR protocol.  The same mix w
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Mutagenesis series: Amplification of a linear strand

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Introduction DNA ligation was successful. D. radiodurans  DNA has been joined together with the DNA of E. coli,  specifically the kanR gene, which has specific primers designed to block the ZWF gene. It was suggested to attempt amplification of this linear strand via PCR.  Procedure A total volume of 20 uL was used containing: 14 uL of a Q5 master mix, 2 uL of each primer (totaling 4 uL) and 2 uL of the genomic DNA. Primer 1 and Primer 6 were used.   Once the sample was prepared, PCR was conducted using a thermocycler with an annealing temperature of about 61 degrees Celsius.  Results  This attempt was unsuccessful.  The picture above depicts the gel that was run on the sample after PCR and there are no DNA bands shown.  There was 5 uL of DNA used as this amount was used previously when the ligation was successful, so a light band was expected to be seen on the gel. The assumption is that because the ligation was completed under certain conditions that this amplification process will n
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Mutagenesis series: 3-way assembly pt 2

By TT -
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  Introduced  The first attempt at creating a linear strand was unsuccessful.  This gel looked like a previous gel from a prior stage of this experiment that had no primers added to the solution.  The assumption is that the primers were not added to the solution so another attempt was made and  primers 1 and 6 were  added to the solution. Procedure   Using the 2-way assembly of the right+middle, the addition of the left fragment was introduced to the solution that consisted of a 5X Q5 master mix, primers 1 and 6, and PCR water.  This process went through Overlap PCR and the Final Fusion steps of the Overlap PCR protocol.  The conditions of the thermocycler were set to the prescribed temperatures and durations that are detailed in the Overlap PCR protocol, which were the same as the step of creating the 2-way strand.  Results Gel of 3-way assembly attempt #2. Bands shown are about 2300 base pairs.   The attempt was successful.  The second attempt at creating a linear strand was successf
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Mutagenesis Series: 3-way assembly

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  Introduced  As it was previously mentioned, this part of the process will be using the 2-way assembly fragments, without clean-up, and adding the missing fragment with the appropriate primers. The procedure that is being followed in the process recommends a clean-up of PCR products, however, the 2-way assembly was successful without a clean-up. The results of this attempt will determine the next step of this series. Procedure   Using the 2-way assembly of the right+middle, the addition of the left fragment was introduced to the solution that consisted of a 5X Q5 master mix, primers 1 and 6, and PCR water.  This process went through Overlap PCR and the Final Fusion steps of the Overlap PCR protocol.  The conditions of the thermocycler were set to the prescribed temperatures and durations that are detailed in the Overlap PCR protocol, which were the same as the step of creating the 2-way strand.  It is important to note that the 2-way assembly fragments have not been through a clean-up
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Mutagenesis series: 3-way assembly planning

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Introduced  The next step of this process will be to attempt a 3-way assembly, by combining left+right+middle fragments. This process will create a linear strand of DNA that will be transformed and taken up by competent Deinococcus radiodurans.    Procedure There are 2 options that may work in the assembly of a 3-way linear strand of DNA.   1.) Using the 2-way assembly of the right+middle or left+middle would allow the addition of just one fragment to the solution such as adding the left fragment to the right+middle assembly.  This process will go through Overlap PCR and the Final Fusion step, to attempt to make the linear strand. 2.) Using the original individual fragments and placing them in one tube. This would also require Overlap PCR and the Final Fusion step.   It is important to note that the 2-way assembly fragments have not been through a clean-up so using the Gel Excised samples may be more beneficial.  The concentration of these samples is very low so it may need to be conce
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Mutagenesis Series: 2-way Assembly

By TT -
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  Introduction Previously, step 1 of the Overlap PCR procedure was conducted. T here were no primers added to these reactions, which led to a gel that showed no bands of DNA because the fragments were not yet fused together.  They were only amplified.  The next step that will be discussed is called the Final Fusion step of this protocol.  This step should result in a 2-way assembly by fusing together the overhang sections created by the primers that will be added.  Procedure This protocol was optimized to eliminate the need for optimizing PCR cycling conditions.  There are no specific temperatures that must be used for the annealing cycle and the temperatures decrease on a gradient of 0.5 degrees Celsius per cycle.   This step was completed in two reactions.  This step required 22.5 uL of Q5 master mix, 4 uL of the step 1 reaction, 1 uL each of primers 1 and 4 (left fragment, tube1), 1u of primers 3 and 6 (right fragment, tube2), and 21.5 uL of PCR water.   These two reactions were put
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Mutagenesis Series: Overlap PCR

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 Introduction PCR is a tool for used to multiply specific fragments of DNA, as an alternative to gene cloning,  that can be used to create gene variations in the laboratory. Overlap extension PCR is a technique that is commonly used for cloning large complex fragments, and fusing two gene fragments together.  In this series, Overlap PCR is used to fuse two gene fragments together.  As mentioned in previous postings.  The KanR gene from E. coli will be used to tag D. radiodurans and block the ZWF gene. The protocol used for this process uses touchdown PCR cycling and is completed in 2 steps. The first step was completed separately.  This posting will go over the 1 step of the Overlap procedure that was completed.  Procedure This protocol was optimized to eliminate the need for optimizing PCR cycling conditions.  There are no specific temperatures that must be used for the annealing cycle and the temperatures decrease on a gradient of 0.5 degrees Celsius per cycle.   This step was comp
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