"
Team:University of Ottawa/16 July 2008
From 2008.igem.org
Contents |
Today on the Lab
Tammy
- Innoculation of transformed X10 E.coli + pDR197::AtCKX2 in LB + AMP.
- For each dilution (PURE, 1:10, 1:100), 2 colonies were chosen to innoculate in 3 ml LM + AMP.
- Control is LB + AMP only.
- Finished Innoculation at 3:oo pm
Chris
- Gel Extraction of AtCRE
- prepared a 0.8% gel and ran the AtCRE sample for 40 minutes at 90 V
- expected bands appeared and the desired one was excised
- gel extraction was performed on the excised sample successfully
- Determining AtCRE Concentration
- measured the absorbancy of AtCRE, resulting in invalid data. It was determined that the blank was performed incorrectly, such that the machine was not zeroed properly.
- the absorbancy of AtCRE was remeasured using a new blank at a 1:10 dilution. The resulting absorbancy was 0.3113 at 260 nm, giving a concentration of about 150 ng/ul.
- Ligation of AtCRE
- AtCRE was ligated with 1 ul ligase, 1 ul ATP, 2 ul DNA template, 1 uL buffer and 5 ul water
- the sample was incubated at 16 C overnight
Matt
- Glycerol Stock
- The BY4742 cells int 597/598 were inoculated once again for glycerol stock tomorrow - this time I sealed the tubes with the parafin.
- Digestion
- Digestion confirmation of PTP2 with NcoI after Gel extraction was successful with correct band sizes confirming that I have the PTP2 product.
- A PCR cleanup was performed on the digestion product of PTP2 digested with BamHI + xhoI, concentrations were low but not low enough that a ligation cannot be performed.
- I still have some pSSA42 digestion product from last time the digestion was performed that I can use for this.
- Ligation
- A ligation was then performed using PTP2 insert in pSSA42 vector using 3:1 mol ratio - I decided to use a little more insert than suggested to ensure success of the reaction.
- Dan was thinking it would probably be easier if the ligation was spiked tomorrow morning with more ATP.
Dan
- Overnight PCR of 0B at 35 cycles
- Was unsucessfull
- PCR of 0B at 35 cycles
- Was redone and did not work again,
- My conclusion is that the higher primer concentration is inhibiting it.
