Jessica Karen Wong/Notebook/2007-6-26

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

Gel from 6/26/07

• 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