User:Howard Boland/Notebook/Art from Synthetic Biology/2010/08/19
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The plates I grew from yesterday User:Howard Boland/Notebook/Art from Synthetic Biology/2010/08/18#Plasmid Transformation (pUA66katE#A1#3 and pUA66katE#A1#4) and User:Howard Boland/Notebook/Art from Synthetic Biology/2010/08/18#Grow pUA66katE#A1 both grew. The colonies on plate pUA66katE#A1#3 looked similar to the original plate pUA66katE#A1 and indicator that this sample contains the insert. I had the digestions already running.
Gel 2%, insert screening
To screen the remaining colonies from pUA66katE#A1 ran overnight digestions on a 2% gel, User:Howard Boland/Notebook/Art from Synthetic Biology/2010/08/18#Overnight Digestion (pUA66katE#A1#3 and pUA66katE#A1#4)
Analysis: None of the colonies screened shows the insert. At this point I have screened all four colonies. I have detected one potential candidate but I did not take a sample and will need to redo a screening for all colonies to verify.
Prepared ladder 100bp
I prepared a new 100bp NEB ladder by adding ( where did I put this protocol?)
Miniprep - overnight growth (67 | pUA66katE#5)
I used 2 bottles out of a total of 6 for miniprep, based on the overnight growth User:Howard Boland/Notebook/Art from Synthetic Biology/2010/08/18#Overnight Growth (pUA66katE and plasmid 67).
For each of the 2 (pUA66katE#5 and p67) remaining bottles:
Digestion - (67 | pUA66katE#5)
I prepared a master mix to digest each of the 30µl plasmid to a final volume of 50µl
Master mix (total volume 40µl)
Gels 1% and 2%, vector extraction and insert screening
Prepare a 2% gel to screen for insert.
Prepare a 1% gel to cut out p67
I prepared 4 bottles of 10ml LB Broth and add 10µl of 50mg/µl Kanamycin stock.
A proper screening of the pUA66katE#A1 plate was performed by labelling each colony. As these colonies had been picked before by swabbing and now regrown I check that none of the smears overlapped.
SAP and Ligation
A key problem with my vector is that there seems to be non-specific binding resulting in self-ligation. This prompted me to return to the use of SAP (Shrimp Alkaline Phosphate) disrupt the binding sites and prevent self-ligation.
Some mistakes were made during this process.
My vector has a concentration of around 15ng/µl, thus in 30µl I have a total of 15ng/µl*30µl = 450ng ~ 0.5µg of DNA. The SAP is used in 1unit/µg of DNA. So I will be using 0.5µl of SAP.
SAP Reaction, final volume will be 50µl.
I did the same reaction for a total of 4 samples from my box.
Four ligations were then setup using all SAP
I setup two reactions in a standard ligation 1:7
All ligations were incubated overnight at 37ºC