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Team:Harvard/vectors/notebook
From 2010.igem.org
abstract lab notebook agrobacteria and plant engineering meet the vectors plant transformation parts & primers references
notebook
06-14-2010 [top]
Started preparation of agrobacterium vector backbones
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
[click to enlarge]06-15-2010 [top]
Continued preparation of agrobacterium vector backbones
Continued preparation of agrobacterium vector backbones
- Re-digested E3, E4, R1, R3 with HindIII and SpeI (Fast Digest Protocol - make 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.
[click to enlarge]
[click to enlarge]
[click to enlarge]06-16-2010 [top]
Completed preparation of agrobacterium vector backbones and prepared expression series and reporter series inserts
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: 60°C
- 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)
[click to enlarge]
[click to enlarge]06-17-2010 [top]
Ligated digested backbones and inserts. Transformed and plated on LB and Kanamycin.
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
- 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 30°C overnight
| Vector | Insert Size | Insert Mass |
| E3 | 600 bp | 13 ng |
| E4 | 600 bp | 13 ng |
| R1 | 2000 bp | 20 ng |
| R3 | 1000 bp | 40 ng |
06-18-2010 [top]
Realized that there was a 1bp error in annealed oligos for O1 and O2 (had sticky end for SpeI site instead of NheI site)
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
- Prepared 50 ug/ml LB+Kan Media.
- 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 37°C for 6 hours
- Centrifuged cultures to produce pellet and removed the liquid
- Stored pellet at -20°C
06-21-2010 [top]
Carried out initial test to verify the identity of plasmid transformed into E. coli
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
[click to enlarge]06-22-2010 [top]
Continued to carry out initial test to verify the identity of plasmid transformed into E. coli
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
[click to enlarge]- 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 37°C overnight
06-23-2010 [top]
Continued to prepare for plasmids to be sequenced
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 37°C for 6 hours
- After 6 hours, had not grown enough, so left cultures overnight at 37ºC
06-24-2010 [top]
Miniprepped V9-V12 cultures. Got yields with enough DNA to send to GeneWiz for sequencing. Primers arrived from Mr. Gene and we will send constructs off for sequencing next week.
Miniprepped V9-V12 cultures. Got yields with enough DNA to send to GeneWiz for sequencing. Primers arrived from Mr. Gene and we will send constructs off for sequencing next week.
Started putting together Open Series constructs
- Annealed oligos synthesized by Mr. Gene to form insert (protocol)
- Ligated with backbone digested last week
- Transformed into TURBO E. Coli and plated onto LB + Kan
06-25-2010 [top]
Continued preparation of Open Series constructs
Continued preparation of Open Series constructs
- Picked colonies from both V7 (O1) and V8 (O2) and set up cultures. Only cultures for V7 grew. Because of the patterns of colonies on the plates, we suspect that the Kanamycin was not properly spread on the plates.
- We had used up all of our remaining digested backbone for O2, so we redid the SacII/SpeI digestion on both vectors with phosphatase, re-ligated, and re-transformed and plated, using a negative control
06-28-2010 [top]
Completed preparation of Open Series construct
Completed preparation of Open Series construct
- Colonies grew for V8 (O2) only. Set up cultures for colonies.
- Miniprepped cultures of both constructs
- Resuspended primers in water
- Mixed required quantities and concentrations of each vector and primer in PCR tubes
- Sent tubes to GeneWiz for sequencing
06-30-2010 [top]
Got back sequencing results (link to sequences)
Got back sequencing results (link to sequences)
- Confirmed V9, V10, V11, V12 - both #5,6 for all. V11 had a single base pair change in the middle of the GusA sequence (bp 6315, T instead of C). This basepair is at the end of a codon, and the change does not change the translation of the sequence.
- V7, V8 had faulty primers - the reverse was not the reverse compliment and the forward was too close to the sequence, so redesigned primers and ordered them.
07-01-2010 [top]
Started to produce more of the V9-V12 vectors
Started to produce more of the V9-V12 vectors
- Set up starter cultures for V9-V12 for maxipreps from colonies from the #5 plate of each construct (in LB+Kan 50ug/ml)
- Poured started cultures in 150mL LB + Kan (50ug/ml)
- Agrobacterium transformation (electroporation)
- Transformed E4 and V10, plus did a control that was shocked without DNA, and a control that was not shocked but had E4 in it.
- Plated on LB+Kan(50ug/ml) + Gent(30ug/ml) + Rif(10ug/ml)
07-06-2010 [top]
- Agrobacterium plates had colonies for E4 and V10 and no growth for the negative controls
- Sequencing V7 and V8 showed they were correct (see sequences)
- Started cultures for maxiprep of V7 and V8
07-12-2010 [top]
- Started putting finished constructs from other teams into vectors
- Digested V9, V10, Miraculin, Brazzein, Miraculin + YFP, Brazzein + YFP with EcoRI and PstI (V9/V10 with TSAP added as well)
- Digested parts appear to be the correct size
- Gel extraction led to low yields, so redid digestions the next day
[click to enlarge]07-13-2010 [top]
- Continued putting finished constructs into vectors
- Digested V9, V10, Miraculin, Brazzein, Miraculin + YFP, Brazzein + YFP, GFP Knockdown 1, GFP Knockdown 3 with EcoRI and PstI (V9/V10 with TSAP added as well)
- Digested parts appear to be the correct size
- Ligated vectors (V9/V10) and inserts to form V21-V32 (see parts page) and transformed into TURBO E. coli
[click to enlarge]07-14-2010 [top]
- Colonies from 7-13-10 transformation were very large and unevenly spread
- Picked two colonies from each ligation and started cultures
07-15-2010 [top]
- Miniprepped cultures for V21-V32 (set up 7-14-10)
- Transformed into Agrobacteria
- Settings: Agr Setting on electroporator
- Amounts: 40 ul electrocompetent cells, 5.5 ul DNA (~100 ng/ul)
- Transformed: V21-V32 #2, V10 as positive control, blank cells as negative control
- Plated transforms onto LB + Kan + Rif + Gent plates
07-16-2010 [top]
- Confirmation digest on V21-V32 #1,2
- All constructs had inserts of the correct size, except V23-2
[click to enlarge]07-19-2010 [top]
- Set up lawn plates on YEB + Kan + Gent + Rif
- V21-V32 except V23, V10, E4 (from prior transform)
- Resuspended each individual colony in 30 ul water, plated 25 ul of resuspension (5 ul kept for sequencing)
07-22-2010 [top]
- Removed lawn plates from incubator in the morning
- E4 did not grow as much, so left it in the incubator (the colony picked to make the plate was older than the others)
