Yi-an, spring 2006
Friday, March 3, 2006
Perry and I transformed the DH5alpha competent cells with pCMV-Tag2a vectors. DH5alpha cells were used because they have higher efficiency in transformation. We placed 50 uL of DH5 alpha cells with 1 uL of the pCMV-Tag2A vectors (which was a 1 uL/uL stock) into a 1.5 mL eppendorf tube. This was repeated for a second transformation (2 pCMV parallel transformations were performed). For the positive control, 2.5 uL of pUC19 was placed into a 1.5 mL eppendorf tube along with 50 uL of DH5alpha competent cells. These cells were placed on ice for 30 minutes. Subsequently, we heat shocked the cells at 42 degrees Celsius for 25 seconds then placed them on ice for 2 additional minutes. SOC medium was incubated and 950 uL were added to each eppendorf tube. The tubes were then placed into the shaker (225 rpm, 37 degrees celsius) for an hour. The protocol used modeled that obtained from the DH5 product information sheet).
We then transformed TOP10 competent cells with hD1g-cloned Topo vectors: SG25, SG35, and F11. We diluted the stock vector 1:10. 5 uL of each of the necessary vectors were added to separate tubes of TOP10 competent cells. 2.5 uL pUC19 was added to one tube of top10 cells to serve as the positive control. Nothing was added to the tube of competent cells - this served as the negative control. The tubes were set on ice for 30 minutes, heat shocked for 25 seconds, set on ice for another 2 minutes, and then placed in the shaker for an hour (225 rpm at 37 degrees Celsius). Perry plated the transformations.
Protocol used for the second step was one i wrote up from mcb100 (included below):
1) Grab an ice bucket and fill it with ice.
2) Obtain 2 tubes from the "One Shot Top 10: Chemically Competent Cells" box found in the bottom shelf of the upper -80 Degree Celsius Freezer.
Obtain: - 1 purple tube - cells - 1 black capped tube - plasmid (for positive control)
3) Label 3 eppendorf tubes and initial:
(+) for positive control with plasmid (-) for negative control with water LIG for ligation DNA (you can use the whole cell vial for this)
4) Pipet 10 - 15 microliters of cells from the purple capped vial into each eppendorf tube.
5) Place 2 microliters of positive (plasmid), (-) water, and ligation DNA into their respective labeled tubes
6) Place the 3 tubes in the ice bucket and set a timer for 15 minutes
7) Place the tubes in the heat plate at 42 degrees Celsius for 30 seconds (Set a timer).
8) Place the tubes back in the ice bucket for 2 minutes (set timer).
9) Pipet 250 microliters of S.O.C. Medium (yellow fluid) into each eppendorf tube.
10) Place the 3 eppendorph tubes in the incubator in the other room (near the computer - to the left) for 45 - 50 minutes.
11) Obtain 3 Amp resistant petri plates (found in the back refrigerator of the other lab room). Label the plates with (+), (-), and LIG.
12) Obtain the 3 incubated vials from the other room. Get glass beads from the back shelf by the window.
13) Pipet the fluid from each vial into their respective petri plates (pipet onto the center of the plate).
14) Pour a few (approximately 10 beads) onto each plate. Cap the plates and shake them side to side [avoid shaking them in a circular manner because this will cause a higher concentration of bacteria to end up in the center of the plate. You want them to be spread out as evenly as possible). Place the used beads in a small beaker, rinse, and place them in the dirty equipment tub for cleaning.
15) Cover the plates and place them in the incubator overnight.
Saturday, March 4, 2006
I came into biolabs tonight and performed another transformation on the DH5alpha competent cells with pCMV-Tag2a vectors as well as another transformation of the TOPO vector (F11, SG25, SG35) with TOP10 competent cells. The same protocol (above) was used for the respective transformations.
Sunday, March 5, 2006
Perry performed an innoculation using the successful transformations from one of the previous transformations.
Tuesday, March 7, 2006
The innoculation yielded very little pellet, thus, we made a Starter culture for pCMV and SG25. 3 mL of LB media was used for each labeled Falcon tube. We placed 1.8 uL of KAN in the pCMV tube and 1.0 uL of Amp in the SG25 tube. A sterile loop was used (note: yellow rods, found in the other room, can be used in place of the sterile loops) to pick a colony from the pCMV transformation plate. The sterile loop end was swooshed in the tube. 10 mL of the SG25 glycerol was added to the respective eppendorf tube. Both tubes were placed in the 37 degree celsius, 225 rpm shaker. The QIAGEN Plasmid Purification Handbook (p.20) states that the incubation period should last 8 hours.
Note to self: for the innoculation, use the LB medium located on the back (far right) shelf (the terrific broth was contaminated).
Protocol for Innoculation: Dilute the starter culture - 200 mL LB, 400 uL culture, 120 uL (KAN - for pCMV) or 200 uL (Amp = for SG25). Grow at 37 degrees Celsius for 12 - 16 hours in the 225 rpm shaker.
Wednesday, March 8, 2006
The innoculations yielded substantial pellets. Alain spun down the pellets and I performed the midiprep.
Midiprep Protocol notes (for future reference):
1) centrifuge the innoculation 6000 rpm for 15 min at 4 degrees Celsius
2) resuspend the bacterial pellet in 4 mL Buffer P1 (use the vortex machine to break up the pellet. Then transfer the pellet to a smaller centrifugation tube (the one we used in mcb100 - clear/transparent tubes)
3) Add 4 mL Buffer P2, mix gently through inverting the tubes 4 - 6X. Incubate at rm temperature for 5 minutes
4) Add 4 mL of chilled P3 buffer, mix gently (invert 4 - 6 X), incubate on ice for 15 minutes.
5) Centrifuge at 13,000 rpm at 30 minutes at 4 degrees Celsius. Remove supernatant. (note to self: mark the edge of the tube which will be facing up in the centrifuge - just so that you can locate the pellet with greater ease later in the procedure).
6) In the meantime, equilibrate the QIAGEN-tip 100 (found in the Midiprep kit) by applying 4 mL of QBT buffer to each column. (i used 4, there is also a new rack - transparent blue - that nullifies the need to set up separate racks - found in room 5088)
7) Apply the supernatant from the centrifugation step to the QIAGEN-tip. After it has flowed through, wash the QIAGEN-tip with 2 X 10 mL QC buffer (also found in the midiprep kit)
8) Get a new set of centrifugation tubes - the clear, opaque ones. Elute the DNA into these tubes with 5 mL QF Buffer.
9) Precipitate the DNA by adding 3.5 mL room temp. isopropanol to the eluted DNA. Mix and centrifuge at 11,000 rpm for 30 minutes at 4 degrees Celsius. Decant the supernatant.
10) Wash the DNA pellet with 2 mL of room temp 70% ethanol (do not disturb pellet). Decant the supernatant (do not disturb the pellet).
11) Air dry the pellet for 5 - 10 minutes. Redissolve the DNA in 250 - 300 uL of distilled H2O.
Performed nanodrops for each of the construct midipreps. Two samples were measured for each midiprep. The results are as follows:
F11: 604.9 ng/uL and 615.2 ng/uL SG25: 229.3 ng/uL and 228.9 ng/uL SG35: 89 ng/uL and 90.4 ng/uL pCMV: 213.5 ng/uL and 212.4 ng/uL
- all the measurements displayed the appropriate 260 nm peak.
Also, Alain showed me how to use the new PCR machine located in room 5096. We programmed "BB2" which stands for BioBricks 2, a simplification due to the inefficiencies of the machine in typing in letters.
The program consists of the following steps:
Step 1: 94 degrees, for 5 minutes
Step 2: 94 degrees, for 30 seconds
Step 3: 50 degrees, for 30 seconds
Step 4: 68 degrees, for 1 minute
Step 5: repeat steps 2 through 5 for 30 cycles
Step 6: hold at 4 degrees, forever (typed in 0 to allow for this)
Step 7: END
Functions of the machine:
- the buttons (<-) and (->) allow you to scroll through options, or input a "space"
- the button (^) allows you to select something that you want
Prepared 4 samples of PCR reactions with the following composition: general formulation: 45 uL PCR supermix, 2 uL distilled water, 1 uL of primer1, 1 uL of primer2, and 1 uL of diluted template.
- Part1: F11 (template) and primers (L27S + L27A)
- Part 2: SG25 (template) and primers (SH3S + GKA)
- Part 3: SG35 (template) and primers (SH3S + GKA)
- Part 4: SG35 (template) and primers (SH3S + I3A)
I diluted the SG25 and F11 in a 1:100 proportion and performed a 1:10 dilution of SG35 (as a result of the aforementioned concentrations obtained for the different constructs.
- Note: All dilutions and templates are located in the large -20 freezer in room 5096 in the clear box on the right side of the second shelf. All primers are located in the green labeled box on the right side of the 2nd shelf in room 5096.
Thursday, March 9, 2006
Retrieved the PCR from last night and ran a mini-gel (2% gel) with the 4 PCR samples and 2 ladders (the 1Kb ladder and the 2 log ladder - both of which can be found in the normal mcb100 project 3 freezer in room 5088)
For the minigel run:
- make sure that all volumes in all wells equals 20 uL. 10 uL of PCR DNA was used for each sample and 10 uL of each ladder was used. 10 uL of water was added in a separate eppendorf tube to mix either ladder or DNA with water. Water was added to all empty wells (20 uL)
- explanation of each of the gel usages (to come): 0.8%, 1.2%, v. 2%
Performed a PCR Purification (protocol):
1. Add 5 volumes of Buffer PB to 1 volume of PCR sample and mix. (200 uL of PB was added)
2. Apply the sample to a QIAquick spin column and centrifuge at 13000 rpm for 1 min. discard flow-through.
3. Replace the column. Add 750 uL PE Buffer and centrifuge for 1 minute. Discard flow through. Replace the column and centrifude for another 1 minute
4. place each column in a clean/new 1.5 mL eppendorf tube. Elute with 30 uL dH2O, let the column stand for 1 minute, centrifuge for 1 minute.
Performed TOPO Cloning:
1) Get the necessary number of tubes (in this case 4 tubes) of Chemically Competent Top10 Cells, place on ice (for use in transformation).
2) get 4 eppendorf tubes (label appropriately #1-4)
3) Create the following mixture: 4 uL Fresh PCR product, 1 uL Salt solution, 1 uL of TOPO vector.
4) Incubate the tubes at room temperature for 5 minutes
5) set on ice then procede to transformation
For the transformation: i followed the proctocol above (taken from my old mcb100 wiki page)
- Diluted the I2-I5-I4 TOPO (1:100)
Performed PCR reaction for the last 5 parts using program BB2:
- Part 5: SG25 (template) and primers (I2S + GKA)
- Part 6: F11 (template) and primers (SH3S + SH3A)
- Part 7: I2-I5-I4/Topo (template) and primers (I2S + I4A)
- Part 8: F11 (template) and primers (GKS + GKA)
- Part 9: I2-I5-I4/Topo (template) and primers (I5S + I4A)
Friday, March 10, 2006
- the transformations that I performed last night did not yield any colonies. There seemed to be a weird bumpy lawn of (something) on the agar; however, they were not colonies.
- Thus, i performed ran an e-gel of the purified PCR DNA (using 10 uL dna and 10 uL water for each well sample). The e-gel showed that there were DNA bands in all expected lanes for the roughly the expected band lengths. The bands however, seemed a bit light/dull.
- Perry performed the cloning, transformation, and purification of parts 5 through 9. Two parts were successful, he re-PCR'd the other 3 parts.
Saturday, March 11, 2006
- Performed another TOPO cloning procedure on Parts 1 through 4. I included a positive control (using pUC 19 cells that were located in the -80 freezer) as well as a negative control (with nuclease free water).
- Performed a transformation on the 6 samples.