IGEM:Harvard/2010/Team Vector: Difference between revisions
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==Vector Diagrams== | ==Vector Diagrams== | ||
[[Image:V5. | [[Image:V5.png|250px]] | ||
==6-14-2010== | ==6-14-2010== |
Revision as of 09:03, 24 June 2010
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)
- Realized that ordered O1, O2 oligos had SacII sticky and rather than HindIII
- 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
- Digested PCR products to prepare products with correct sticky ends (two digests per vector)
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.