IGEM:Harvard/2010/Team Vector

Vector Diagrams

6-14-2010

Started preparation of agrobacterium vector backbones

• Digested O1, O2, E3, E4, R1, R3 with HindIII and SpeI (Fast Digest Protocol - make link)
• Ran digests on 1% agarose gel (protocol link)
• Digest successful for O1, O2, and R1 but not for E3, E4, and R3 (reason unknown)
• 
• Cut out backbone (top) bands from O1, O2, R1 and saved them at 4°C

6-15-2010

Continued preparation of agrobacterium vector backbones

• Re-digested E3, E4, R1, R3 with HindIII and SpeI (Fast Digest Protocol - make link)
  • Ran digests on 1% agarose gel (protocol link)
  • Digest successful for all reactions
  • 
  • Cut out backbone (top) bands for E3, E4, R3 from the gel
• Realized that ordered O1, O2 oligos had SacII sticky and rather than HindIII
  • Re-digested O1, O2 with SacII and SpeI to prepare backbone with the correct sticky ends (regular digest protocol, buffer 4)
  • Ran digests on 1% agarose gel (protocol link)
  • Digest successful for both reactions
  • 
  • Cut out backbone bands for both reactions
• Annealed open series insert oligos (synthesized by Mr. Gene) (protocol link - karmella's)
• Gel purified bands for all reactions (protocol link)
• Realized that backbones needed to be dephosphorylated before ligation, so redid digestions with phosphatase.
  • Digested O1, O2 with SacII and SpeI, with TSAP (phosphatase) added (slow digest protocol, buffer 4, 30 ul reactions). Did two digest reactions for each vector to increase yields.
  • Digested E3, E4, R1, R3 with HindIII and SpeI, with TSAP added (fast digest protocol, 30 ul reactions). Did two digest reactions for each vector to increase yields.
  • Ran digests on 1% agarose gel (protocol link)
  • Digest successful for all reactions
  • 
  • Cut out backbone (top) bands for each reaction, keeping the two digests of each vector in the same eppendorf tube.

6-16-2010

Completed preparation of agrobacterium vector backbones and prepared expression series and reporter series inserts

• Gel purified digest slices from previous day (protocol link)
• Ran a PCR reaction on E3, E4, R1, R3 vectors with primers synthesized by Mr. Gene (PCR phusion protocol) to generate inserts
  • Melting Temperature: 60ºC
  • Elongation Time: E3, E4, R3 (~0.6 kb) - 15 seconds; R1 (~2 kb) - 30 seconds
  • Ran part of each reaction on 1% agarose gel to determine if correct products were formed
  • PCR reactions were successful
  • 
  • PCR purified remainder of each reaction
• Digested PCR products to prepare products with correct sticky ends (two digests per vector)
  • Digested with HindIII and SpeI (Fast Digest Protocol, 20 ul reactions)
  • Ran on 1% agarose gel (protocol)
  • 
  • Cut out insert (top) bands for each reaction, keeping the two digests of each product in the same eppendorf tube.
  • Gel purified products (protocol)

6-17-2010

Ligated digested backbones and inserts. Transformed and plated on LB and Kanamycin.

• Prepared LB plates with 50 ug/ml kanamycin (15 ug of kanamycin per plate)
• Prepared 50 ug/ml LB+Kan Media.
• Ligated backbones and inserts of expression and reporter series vectors (ligation protocol)
  • Used 50 ng of digested, dephosphorylated backbone DNA
  • Used roughly 3x molar excess of digested vector
    • Mass of Insert = [Size of Insert][Molar excess][Mass of Vector]/[Size of Vector]
    • Size of Agrobacterium vector backbone: ~7 kb

Vector	Insert Size	Insert Mass
E3	600 bp	13 ng
E3	600 bp	13 ng
R1	2000 bp	20 ng
R3	1000 bp	40 ng

• Transformed ligation reactions into TURBO e. coli cells (protocol)
  • Transformed 5 ul of reaction mix into 15 ul of cells
  • Plated onto LB + Kan plates, left at 30ºC overnight

6-18-2010

Realized that there was a 1bp error in annealed oligos for O1 and O2 (had sticky end for SpeI site instead of NheI site)

• Ordered new oligos with corrected bp

Started to obtain E3, E4, R1 and R3 plasmids from E. coli

• Picked four colonies of E. coli from the E3, E4, R1 and R3 plates and made a culture from each in LB and Kanamycin
• Left cultures at 37ºC for 6 hours
• Centrifuged cultures to produce pellet and removed the liquid
• Stored pellet at -20ºC

6-21-2010

Carried out initial test to verify the identity of plasmid transformed into E. coli

• Carried out miniprep to obtain plasmids from cells (protocol link)
• Digested plasmids with EcoI and HindIII (protocol link)
• Ran digests on 1% agarose gel (protocol link)
• 
• Ladder was unclear so needed to repeat
• Measured concentration of miniprep product
  • Concentrations were low (<44ng/uL) so needed to redo miniprep
• Picked new colonies (2 from each plate) and grew cultures overnight

6-22-2010

Continued to carry out initial test to verify the identity of plasmid transformed into E. coli

• Carried out miniprep to obtain plasmids from cells (protocol link)
• Digested plasmids with EcoI and HindIII (protocol link)
• Ran digests on 1% agarose gel (protocol link)
• 
• Larger bands were observed, suggesting the correct backbones were present
• Smaller bands were observed, suggesting the correct inserts were present

Started to prepare for plasmids to be sequenced

• Required more plasmids
  • Transformed plasmids back into E. coli using heat shock (carried out twice for each of V9-12) (see protocol)
  • Added Kanamycin to LB plates
  • Spread E. coli cells onto LB + Kanamycin plates
  • Left plates in 37ºC overnight

6-23-2010

Continued to prepare for plasmids to be sequenced

• Picked 3 colonies from each of the V9, V10 and V12 plates and 4 colonies from each of the V11 plates
• Left at 37ºC for 6 hours
• After 6 hours, had not grown enough, so left cultures overnight at 37ºC

6-24-2010

Miniprepped V9-V12 cultures. Got yields with enough DNA to send to GeneWiz for sequencing. Primers will not arrive until next week.