Jessica Karen Wong/Notebook/2007-6-26
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To Do
- Take out PCR and Digest
- PCR clean up
- Run analytic gel on PCR of I
- Make new Tet plates
- Re-plate devices
- Miniprep and digest 3K3
- Re-order T & E primers?
- PCR E and I on a gradient
Stopped PCR and heat shocked digest
Poured new Chlor LB plates
Nishant miniprepped and digested 3K3
Gel of overnight PCR
- Ran an analytic gel on I2055 PCR product see protocol
- No visible product
- Will PCR on a temperature gradient
PCR Cleanup
- PCR purified the 3 sucessful backbone digests (1AC3, 1AK3, 1AT3) and the PCR of I2055
- Had 50ul of each digest and 90ul of the PCR
Designing Primers
- New T9002 Reverse that matches only GFP TCAGCCAT ATGCAT CCATGCCATGTGTAATCCCAG
- Melting Temp 55.0
- Original T9002_F CTTAGTAG CAATTG TCCCTATCAGTGATAGAGATTGACATC has melting temp 53.9
Bold is the tail, italics is the restriction site.
- New Longer I2055
- Fwd- TATAAACGCAGAAAGGCCCACCC
- Original I2055
- Fwd- CTTAGTAG + CAATTG + tccctatcagtgatagagattgacatc
- Rev- TCAGCGAT + ATGCAT + TATAAACGCAGAAAGGCCCAC
Gradient PCR
- Did a 10ul analytic PCR of E0240 and I2055 on a gradient from 49 to 60
- E0240F melting temp - 53.1
- E0240R - 53.6
- I2055F - 53.9
- I2055R - 53.6
- Temperature in each of the 12 columns: 1st - 49, 2nd- 49.3, 3rd- 49.9, 4th-50.8, 5th-52.1, 6th- 53.7, 7th- 55.6, 8th- 57.2, 9th- 58.3, 10th- 59.2, 11th- 59.8, 12th- 60
Plated Devices
- From yesterday's overnight plates only saw colonies on blue C (RBS tester on Chlor)
- Spun down the rest of the cultures at 4k for 4 min
- Removed most of supernatant and resuspended in the remaining 100ul of LB
- Plated both devices on both Tet and Chlor
Gel of Gradient PCR
- Ran a 2 row 20-lane analytic gel
- 1. E0240 _ L _ _ 1 2 3 ...12 _ L _ _
- 2. I2055 _ _ L _ 1 2 3 ...12 _ _ L _
- E0240 has a bright band of reasonable length (slightly <1k)
- I2055 is very blurry and the brightest band is 3x longer than the part - very strange