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Team:University of Lethbridge/Notebook/GeneralLabAugust
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Objective: Run a gel to confirm that appropriate inserts were amplified from the PCRs and do a gel extraction of the inserts to prepare them for the biobrick format. | Objective: Run a gel to confirm that appropriate inserts were amplified from the PCRs and do a gel extraction of the inserts to prepare them for the biobrick format. | ||
| - | -Could not obtain a picture of the gel (1% agarose) of the digested pSB1A7 and the recently amplified CheZ gene because the camera would not turn on. | + | -Could not obtain a picture of the gel (1% agarose) of the digested pSB1A7 and the recently amplified CheZ gene (15 uL x 3 wells) because the camera would not turn on. |
The CheZ gene appeared at the correct size (~700 bp). | The CheZ gene appeared at the correct size (~700 bp). | ||
| - | -Did a gel extraction of pSB1A7 and CheZ from that gel with the Qiagen MiniElute Gel Extraction kit. | + | -Did a gel extraction of pSB1A7 and CheZ from that gel with the Qiagen MiniElute Gel Extraction kit. Final volume of each was 10 uL. |
| - | -Ran another 1% agarose gel to confirm that the gel extraction was successful. Bands appeared at appropriate sizes. The concentrations of pSB1A7 | + | -Ran another 1% agarose gel to confirm that the gel extraction was successful. Ran 1 uL of each sample. Bands appeared at appropriate sizes. The concentrations of pSB1A7 |
| - | and CheZ were estimated to be 25 ng/uL and 80 ng/ | + | and CheZ were estimated to be 25 ng/uL and 80 ng/uL, respectively. |
Next step: Digest both pSB1A7 and CheZ with phosphatase. A restriction digest will be performed at a later date on CheZ to prepare it for insertion. | Next step: Digest both pSB1A7 and CheZ with phosphatase. A restriction digest will be performed at a later date on CheZ to prepare it for insertion. | ||
Revision as of 21:38, 23 August 2008
Contents |
August 13, 2008
Nathan Puhl and Sebastian
Verified and quantified the LacI and DT restriction digest products.
Set up a Ligase experiment for LacI and DT
- 1.3 uL vector(DT) - 8 uL insert(LacI) - 2 uL 10x Ligase buffer - 1.5 uL Ligase - 7.2 uL ddH2O - 20 uL total volume
August 14, 15
Nathan Puhl, Munima, Selina
Poured 61 LB + Amp plates. Stored in iGEM 4 C fridge.
August 16, 2008
Roxanne
Used Qiagen Plasmid MiniPrep Kit to Plasmid Prep Last Year's Biobrick Parts that were Incubated overnight.
Ran Plasmids in a 1% Agarose Gel at 100 V for 30 minutes.
August 18
Christa, Nathan Puhl, Munima
Ran a colony PCR of riboswitch extracted from gel done by Nathan and Roxanne on a previous day. Roxanne will remove the PCR tube from the thermocycler in the morning.
August 19, 2008
Christa, Munima, Sebastian
Did a plasmid prep on pSB1A7 using QIAprep Spin Miniprep Kit. Stocked 4- 50uL of pSB1A7 and stored in iGEM -20 C.
Sebastian, Nathan Puhl
Ran products from plasmid prep (pSB1A7 x 4 samples) on 1% agarose gel for 27 minutes.
-Lane 1 - 1 kb GeneRuler ladder (2 uL) -Lane 2 -6 pSB1A7 (3 uL) + 6x loading dye (2 uL); Mixed up what sample was in Lane 4, so Lanes 5 and 6 were run.
Conclusion: The bands appeared to be at the correct size for pSB1A7.
August 22, 2008
Christa, Munima, Nathan Puhl, Roxanne
Objective: Run a gel to confirm that appropriate inserts were amplified from the PCRs and do a gel extraction of the inserts to prepare them for the biobrick format.
-Could not obtain a picture of the gel (1% agarose) of the digested pSB1A7 and the recently amplified CheZ gene (15 uL x 3 wells) because the camera would not turn on. The CheZ gene appeared at the correct size (~700 bp). -Did a gel extraction of pSB1A7 and CheZ from that gel with the Qiagen MiniElute Gel Extraction kit. Final volume of each was 10 uL. -Ran another 1% agarose gel to confirm that the gel extraction was successful. Ran 1 uL of each sample. Bands appeared at appropriate sizes. The concentrations of pSB1A7 and CheZ were estimated to be 25 ng/uL and 80 ng/uL, respectively.
Next step: Digest both pSB1A7 and CheZ with phosphatase. A restriction digest will be performed at a later date on CheZ to prepare it for insertion.
