S-STEM Scholar Aland Ali's Blog

 Introduction In the last GelRed agarose gel containing PCR of genomic bead-beading and 7kb plasmid extraction, smears were detected with the PCR reactions whereas a band at 7kb was seen with one of the other plasmid extractions. This specific sample will be used in a long amplification PCR reaction to prepare for Gibson assembly if a clear band it detected at 7kb on an agarose gel. Furthermore, the GelRed proved to be a sensitive and introspective tool to replace UView dye.  After PCR, 7kb samples and CmR samples from earlier may need to be cleaned up using the Monarch PCR & DNA Cleanup Kit (5ug) if any genomic DNA is present. This is because Gibson assembly requires a clean vector and insert for calculating pM ratios.  Methods To run PCR reactions, two 25ul reactions were prepared with 20ul of Taq1 master mix containing 3M MgCl. MgCl is added to promote specific binding of polymerase to the template. 2 ul of forward and reverse primers for the 7kb plasmid were then added. Lastly,

 Introduction  Recently, a heat gradient from 50 °C-66 °C   on the 7kb plasmid from D.aquaticus was ran and contained no visible banding. Due to an empty agarose gel, this may reveal that the template we're using ( boiled cells ) doesn't contain the 7kb plasmid. This would mean our primers aren't able to bind appropriately. Another possibility is that our annealing temperature. Therefore, the alternative bead-beating template will be used in a PCR reaction and run on the more sensitive GelRed agarose gel.  Additionally, a new lysing protocol involving proteinase k and lysozyme will be used to try to extract 7kb plasmid. A theory to test is that we've obtained 7kb plasmid and hadn't seen bands due to the previous dye needing >500ng of template DNA. This new GelRed should only need 5-50ng of DNA to bind visibly.  Methods To run LongAmp PCR products, PCR settings were set to the following: initial denaturing was set to 4 minutes at 94 °C, denaturing for 30 seconds a