4/29-5/3 PCR Using Q5 Polymerase and PCR Cleanup

 We ran PCR on the Gibson product that we had gotten from last week. In this PCR we had used the Q5 Polymerase since it would give us more yield that the usual Taq polymerase that we use. For this PCR we had ran it on the PCR product that we had gotten from last week and on the original Gibson Assembly prior to any PCR. On top of that we ran Long Amp PCR on our PRAD 1 Plasmids again to give us more sample of PRAD 1 vector to work with when attempting Gibson Assembly on the entire plasmid. The next day we ran one of the bigger gels, the ones that have around 15 wells. We needed to use this one since we had 9 wells that we had to fill since we had 4 Gibson PCR products, 4 Long Amp PCR products, and the 1kb ladder. This gel did take a little longer due to the size difference, however once it was done we viewed it under UV light immediately. What we saw was that the PCR on the already PCR’d Gibson products appeared to completely disappear which was a little worrying to us at first. The PCR on the original Gibson Assembly did appear to have a much more prevalent band than what we had seen in the previous week. The brighter bands is most likely due to the the Q5 Polymerase being used. However, this was ultimately a really good sign as we had even more 3-Way Gibson Assembly and with a higher yield of the assembly fragments. 

Well 1: 1KB Ladder, Well 2 &3: PCR’d Gibson Assembly, Well 4&5: Original Gibson Assembly, 
Well 6-9 Long Amp PCR 
Due to the prominence of the extra bands that we can see in the Long Amp PCR fragments we had decided to do PCR Cleanup Kit which actually proved to be very useful and made the clarity of the fragment and didn’t impact the size of the fragment too badly. Overall, the week was a really good step forward for the Gibson Assembly project now that we have some good 3-Way Fragments and clean PRAD 1 vector so we can move forward to fully assemble the plasmid. 

Source: 
Q5 High-Fidelity DNA Polymerase: https://www.neb.com/en-us/products/m0491-q5-high-fidelity-dna-polymerase#:~:text=Q5%C2%AE%20High%2DFidelity%20DNA%20Polymerase%20is%20a%20high%2Dfidelity,to%20support%20robust%20DNA%20amplification. 


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