Yi-An, Fall 2005
my contact information
Dorm: Leverett A-21
cell: 443.286.3377
email: yko@fas.harvard.edu
aim: yksmiles
hope some of this helps :)
Note: Titles in the Table of Contents that are written in all CAPS are protocols. Titles displayed in normal font are results, pictures, summaries of what i did.
BACTERIAL INNOCULATION PROTOCOL
- step into the time machine*
October 7, 2005
1. Collect materials : 3 microliters of Ampicilin (for each tube), 3 milliliters of LB (for each tube), an ice bucket, 6 Falcon tubes, 6 minigrip sterile loops, pipets, a reporter (J06702 mCherry), a promoter (R0051), and constructs (J06750, J06602, J06603, J06611).
Note: make sure a flame/bunsen burner is running to ensure sterile workspace.
2. Label the Falcon tubes with the promoter/reporter/construct that will be placed in it
3. Measure 20 mL of LB into a large tube. Use the electric pipet with the 25 mL pipetting tube.
4. Use the electric pipet (new tube - 10 mL disposable tube) and transfer 3 mL of LB to each Falcon tube.
5. Use a manual pipet and transfer 3 microliters of Amp to each tube (the ampicilin is stored in a clear eppendorf tube in one of the 4 degree Celcius refrigerators - if you are looking for the Kan solution, it can be found in the right-most fridge in the back of the other room).
6. Use the sterile loops and swirl the respective construct/reporter/promoter in the appropriate Falcon tubes.
7. Cap the Falcon tubes tightly and place them in the shaker overnight.
--Yi-An 13:51, 7 October 2005 (EDT)
MINIPREP PROTOCOL
October 8, 2005
1) Gather overnight bacteria innoculation; Label 6 eppendorf tubes with your initials (on the cap) and the appropriate reporter/promoter/construct name on the side. Label 6 spin columns with teh same info on the blue column.
2) Transfer 1500 microliters of each innoculation into their respective eppendorf tubes (2 X 750 microliters). Cap, then centrifuge at 8000 - 14000 rpm for 2 minutes {remember: balance the tubes across from each other).
3) Use the suction device (normal blue pipet tip) to remove the supernatant.
4) Add 250 microliters of P1 buffer to each tube, then use the vortex to dissolve the pellet.
5) Add 250 microliters of P2 buffer; After adding 250 microliters P2 to the first pellet, set the time for 5 minutes, then finish adding P2 to the other tubes.
6)After 5 minutes has passed, add 350 microliters of N3 buffer to each tube.
7) Cap the eppendorf tubes and centrifuge for 10 minutes at 13000 - 14000 rpm.
8) Label another set of eppendorf tubes
9) Set the 1000 microliter pipet to 900 - 1000 microliters and transfer each supernatant to their respective spin columns (be careful not to disturb the cellular debris on the tube sides). Dispose of the Falcon tubes and centrifuge for 60 seconds at 13000 - 14000 rpm. Suction the supernatant.
10) Add 750 microliters of PE Wash Buffer. Centrifuge spin columns for 60 seconds at 13000- 14000 rpm. Suction the supernatant.
11) Centrifuge again for 60 seconds at 13000 - 14000 rpm. Transfer the blue columns into the new set of eppendorf tubes.
12) Add 50 microliters of water to each tube (Aim at the white filter). Centrifuge for 1 minute.
--Yi-An 13:59, 8 October 2005 (EDT)
CHECKING FOR DNA
(was not performed on the first trial, where my DNA disappeared; was performed for the second miniprep on 10/10)
October 8, 2005
1) Prepare DNA for gel loading:
for each reporter/promoter/construct (6 total) create a set of labeled and initialed eppendorf tubes. Fill each tube with the respective DNA (5 microliters), 13 microliters of water, and 2 microliters of loading dye.
2) Obtain the red E-Gel apparatus and the gel (0.8% Agarose Gel)
3) If necessary, cover the right side electrodes with little pieces of aluminum foil to enhance conductivity. Snap the gel in place ( the apparatus should light up).
4) Press the lieft button to pre-run the Gel (there should be a blinking green light for roughly 2 minutes. When time is up, the apparatus will beep). This pre-run will cause the ethidium bromide to move towards the top of the gel (the opposite direction of DNA movement). Load your DNA (20 microliters in each well).
5) Then, press the right-top button. The Gel will run for 30 minutes.
6) Check for DNA on the computer/trans UV machine in the other room.
--Yi-An 13:59, 8 October 2005 (EDT)
DNA DIGEST PROTOCOL
this digest is performed for the reporter and promoter
October 8, 2005
1) Label 2 eppendorf tubes: one for the promoter and one for the reporter
2) collect the following for each tube:
- 3 microliters RB#2 - 15 microliters DNA - 0.3 microliters BSA - 0.5 microliters Restriction Enzyme (RE) #1 - 0.5 microliters RE #2 - 10.7 microliters water
TOTAL: 30 microliters
Note: RB#2, BSA, and RE's are located in teh long Neon yellow container in the other room. Use a blue mini caddy to transport the vials and to keep them cool.
3) Place the digest tubes in the incubator overnight (37 degrees Celcius).
HOW TO MAKE AGAROSE GELS
(10/8)
1) Weight 1 gram of Agarose on a weighing paper - large (found in the drawer below the microwave) using the the electronic balance. (you can use a spatula/scooper to measure - this can be found in the second drawer) Note: If you accidentally place too much agarose onto the balance, DO NOT return the excess to the original container - SIMPLY DISCARD
2) Place the 1 gram of Agarose into a screw-scapped plastic Erlenmeyer flask (found on the 4th shelf from the top of the chalkboard cabinets).
3) Use a graduated cylinder (found hanging from the racks on/near the back sink) and measure out 100 mL of TBE buffer (1X) (found on the table near the cell culture room). When you are finished, rinse the cylinder with distilled water. - FOR A 1 PERSON GEL: only 1 g Agarose is used. Only place part of the 100 mL TBE into Erlenmeyer then heat. Use the remaining buffer as a means for cooling. -FOR A 2 PERSON GEL: Use 2 g agarose. Add the 100 mL to the Erlenmeyer
4) Heat the Erlenmeyer flask (after capping it) using the microwave [approximately 1 minute 15 seconders - initial interval 30 seconds until the liquid begins to boil - try to get it so there is no foamy substance near the top).
5) Add 5 microliters of ethidium bromide (make sure to wear gloves when doing this). Shake the Erlenmeyer flask.
6) Set up the gel apparatus:
a) Use the leveling/plumb device to make sure that the apparatus is horizontal [this can be found on the desk near the cell culture room]. b) Adjust the apparatus using the two knobs situated at the top of the apparatus (from an aerial view) c)Place the plastic clear loading plate into the apparatus d) Clamp the plate into place by turning the large centered knob (located on one of the well walls) until it sinks downwards and locks/tightens. e) place the comb into the appropriate grooved notch f) pour the agarose mixture into the rectangular space g) alow the gel to cool for 15 - 20 minutes (gel should have a milky appearance when it solidifies) h) Wash out the used erlenmeyer flas and place it in the dirty equipment tub.
IN THE MEANTIME...
7) Take the incubated DNA digest and place the eppendorf tubes (2 - 1 promoter, 1 reporter) into the centrifuge for 1 minute at 13000 rpm.
8) Add 3 microliters of 50% Glycerol in TBE + Bromophenol Blue to each eppendorf tube [this is loading dye which can be found in the other lab room or on the table next to the cell culture room].
9) Get a ladder {from the desk next to the cell culture room). It should be either 100 bp or 1 KB ladder (depending on the length of the digested DNA)
IF NO LADDER IS AVAILABLE: - Go to teh back freezer of the other lab room to get some - you must perform a dilution with buffer - a) measure how much of each ladder you have using a pipet. record the quantity. - b) Figure out the total quantity 10X - loading dye is 10X, thus calculate how much is needed - c) find the difference between the total volunme and the sume of the ladder and loading dye volume = volume of buffer
volume of ladder + volume of loading dye + volume of buffer = total volume Examples: 100 bp ladder 1 Kb ladder
17 microliters ladder 27 microliters ladder
170 microliters total 270 microliteres total
17 microliters of loading dye 27 microliters of loading dye
136 microliters of 1X TBE 226 microliters of 1X TBE
BACK TO THE GEL...
10) When the gel has turned a milky color, you know the gel has solidified. Unclamp the gel by turning the clamping knob in the opposite direction. Slowly (with 2 hands) pull the retractable wall (with the clamp) away from teh gel plate.
11) With 2 hands, gently remove the comb
12) Lift the gel plate and click it into place in teh gel electrophoresis apparatus (make sure that the plate is secured)[make sure the gell wells are closest to the black electrode].
13) Get Ethidium bromide (from the back shelf) and place 10 microliters of it in EACH of the gel electrophoresis apparatus wells (NOT THE GEL WELLS)
14) Fill the apparatus wells with 1X TBE until the gel is just covered with a thin layer of TBW (use a side view of the apparatus to ascertain how high the buffer level is).
15) Note where you plan to place each ladder or DNA digest.
---- ---- ---- ----
1 2 3 4
for my experiment: 1. 1 Kb ladder 2. Promoter: R0051 3. Reporter: J06702 mCherry 4. 100 bp ladder
16) Pipet 10 microliters of each DNA ladder into their respective gel wells. Pipet 33 microliters of each DNA Digest into their respective gel wells.
17) Pipetting tips:
a) DO NOT reuse tips. Discard after each use.
b) When pipetting, make sure that there are NO AIR BUBBLES in the pipetted liquid (ladder or digest)
NOTE: Air bubbles act like vortexes when released into a gel well and will cause the injected DNA to spill out of the well.
c) to avoid a re-uptake of DNA from teh gel well, do not release the pipetting button until after you remove the pipet tip from the gel.
18) Place the cover onto the apparatus matching the color electrodes (red-red, black-black).
19) Plug the wires into the POWER PAC machines (R-R, B-B)
20) Set the machine at 100V. PRess RUN (a button that looks like a person in motion). Monitor the Gel run for about 45 - 50 minutes.
21) When the bands of DNA get close to the other edge of the gel, STOP the gel from running and change the voltage setting to 10 V. Run the gel to completion (reaches near-edge - about 2 cm from edge).
--Yi-An 13:59, 8 October 2005 (EDT)
LOOKING AT THE DNA
23) Go into the other lab room and find the gel imaging machine on your far right.
24) Open the bottom drawer of the machine (if the surface is dirty, Clean it with distilled water on a Kimwipe). Plce the gel plate on the surface and close the drawer [make sure all machine doors are closed - otherwise the UV light will not turn on]
25) Go to the computer and open the program "Quantity One" [this program has a red, streaky profile icon - on the program bar]
26) After the program has opened, Go to file --> "Gel Doc EQ"
27) Press the "TRANS UV" button on the machine. Check to see if your ladders and DNA bands are present
28) Regardless of result, click "Video Print" to record results.
29) Save under your folder into the computer. You can also save the image as a JPEG. Go to file --> Export to JPEG --> maximize quality --> press "Export" [Note: these pictures cannot be accessed by any other computer in the lab. You MUST save as a JPEG and open Safari --> webmail --> send the image to yourself]
--Yi-An 13:59, 8 October 2005 (EDT)
Results from Trial 1
(Innoculation/Miniprep/Digestion/Gel Electrophoresis)
VIDEO PRINT 1 Description: The Bands from wells #1 and #4 correspond to the 1 Kb ladder and the 100 bp ladder respectively. Wells #2 and #3 do not exhibit any of the expected DNA bands. A new test (backtracking) was performed to ascertain the source of the problem (either digestion or absence of DNA).
Trial 2 Begins
October 9, 2005
- I restarted the bacterial innoculation due to the absence of DNA on the gel. I followed the previously documented protocol.
October 10, 2005
- I performed a miniprep and revised the protocol. - I performed an E-gel to check for the presence of DNA immediately after finishing the miniprep. - Lastly, I performed a DNA Digestion
--Yi-An 13:59, 10 October 2005 (EDT)
E-Gel Video Print 2
---- ---- ---- ---- ---- ---- ----
1 2 3 4 5 6
1. Reporter J06702 mCherry
2. Promoter R0051
3. J06750 (construct)
4. J06602 (construct)
5. J06603 (construct)
6. J06611 (construct)
Promoter displays a weak signal while the reporter and 3 constructs have clearly visible bands.
TBE DILUTION
October 11, 2005
If TBE runs out...
1) Use the 10X TBE (found in the back of the lab on the shelf nearest to the window)
2) Measure 200 mL of 10X TBW using a graduated cylinder
3) Measure 1800 mL of distilled water (found in the electric dispenser (above the back sink) using the 2L graduated cylinder
4) Add both TBE and the distilled water to the 1X TBE container
5) Rinse out the graduated cylinder and put them back on the hanging rack.
--Yi-An 13:59, 11 October 2005 (EDT)
Trial 2 Continues
October 11, 2005
VIDEO PRINT 3
I made a gel and ran it with the following well setup:
---- ---- ---- ---- ---- ---- ---- ---- ---- ---- ---- ----
1 2 3 4 5 6 7 8 9 10
1. 1 Kb ladder
2. Digested J06702 mCherry (Reporter)
3. Digested R0051 (Promoter)
4. Uncut J06702 mCherry
5. Uncut R0051
6. Uncut J06750
7. Uncut J06602
8. Uncut J06603
9. Uncut J06611
10. 100 bp ladder
IN THE MEANTIME...
Practice Transformation
1) Grab an Ice Bucket and fill it with ice.
2) Grab a tube from the -80 degree Celsius freezer - One Shot Top 10 Chemically Competent Cells [found in a red and white box in the top -80 degree Celsius freezer - bottom shelf]. Place the tube in the ice bucket.
3) Add 0.2 microliters of DNA to the "One Shot" tube [Make sure you can see DNA in the pipet tip.
4) Microfuge (little black circular device that looks liek a 'mini' centrifuge with a clear top) Note: be careful, the cap is hard to reopen.
5) Plac ethe tube ack into the ice bucket. Leave the tube in the bucket for 20 minutes.
6) Heat shock the bacteria for 30 seconds at 42 degrees Celsius and then place the tubes back on ice for 2 minutes.
Perry plated his and my transformations (thanks a bunch! :))
Note: this PRACTICE Transformation was with the miniprep DNA - thus these are PURE PLASMIDS and have much greater efficiency than the LIGATION DNA.
... TO BE CONTINUED...
--Yi-An 13:59, 11 October 2005 (EDT)
QIAQUICK GEL EXTRACTION KIT PROTOCOL
using a microfuge
October 12, 2005
1) Excise the DNA fragment from the agarose gel with a clean, sharp scalpel (minimize the size of the gel by removing the extra agarose).
This step is performed on the pull-out tray of the TRANS UV - gel imaging machine. Make sure that there is a protective piece of plastic that is inserted into the edge of the drawer. Turn off the lights then PRESS the TRANS UV button (while standing behind the protective piece). When you finish cutting, prepare two labeled eppendorf tubes and place the agarose chunks into their respective tubes. Note: the double band well is the reporter - cut out the bottom band. the other single band is the promoter - cut that out and put it into a tube. When you finish, turn off the TRANS UV light and place the used-blade into the red sharps disposal container behind the microscope (to the right of the computer).
2) [optional] weigh the gel slice in a colorless tube.
3) Add three volumes of Buffer QG to 1 volume of gel (100 microliters). For small pieces, add 300 microliters of QG to each tube.
4) Incubate at 50 degrees Celsius for 10 minutes (or until the gel slice has completely dissolved). To help dissolve gel, mix by vortexing the tube every 2 - 3 minutes during the incubation.
The 50 degree Celsius incubation heat plates are in the second to last lab space from the back. Place water in teh wells in order to improve heat transfer to your eppendorph contents. Set a timer for 10 minutes.
5) After the gel has dissolved, check that the color of the mixture is yellow.
- if the color is orange or violet, add 10 microliters of 3M sodium acetate, pH 5.0 and mix. The color will turn yellow. - DNA absorption is only efficient at pH less than or equal to 7.5 and orange/violet is a higher pH
6) Add 100 microliters of isopropanol (isopropyl alcohol) - [located in the cabinets under the hood in the back, closest to the window]
7) Get purple QIAquick Spin columns (2).
8) Apply the sample to the spin column and centrifuge for 1 minute at 13000 rpm. Discard the flow-through then place the column back.
9) Add 0.5 mL of Buffer QG to the column and centrifuge for 1 minute at 13000 rpm. Discard flow-through.
10) To wash, add 0.75 mL of PE Buffer to the column and centrifuge for 1 minute at 13000 rpm. Discard the flow-through. Centrifuge for an additional 1 minute at 13000 rpm.
11) Place the QIAquick Spin column into a clean 1.5 mL microcentrifuge tube
12) To elute DNA, Add 50 microliters of water (or EB Buffer) to the center of the QIA membrane and centrifuge for 1 minute (for increased concentration, only add 30 microliters water)
--Yi-An 13:59, 12 October 2005 (EDT)
DNA LIGATION PROTOCOL
October 12, 2005
1) Obtain the Rapid DNA Ligation Kit [white box located on the top shelf of the large refrigerator in the other room] - protocol is in the box
2) If you have more than 10 microliters of promoter and reporter (both with visible gel bands)[roughly 1:3 ratio of promoter to reporter - 7 microliters of reporter and 3 microliters of promoter]. Remember to keep the T4 DNA ligase on ice!
If you have less than 10 microliters, use the 1x concentration DNA dilution buffer to reach a total volume of 10 microliters.
3) Mix the T4 DNA ligase thoroughly. Add 10 microliters of T4 DNA ligation Buffern (vial 1) to the reaction vial, mix thoroughly via pipetting.
4) Add 1 microliter of T4 DNA ligase (vial 3). Mix Thoroughly.
5) Incubate for 5 minutes at 15 - 25 degrees Celsius (room temp). Set a timer.
--Yi-An 13:59, 12 October 2005 (EDT)
TRANSFORMATION
October 12, 2005
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.
PRACTICE TRANSFORMATION (pure plasmid) CONTINUED...
1) Retrieve the overnight incubated plates. Check for colonies
- Amp resistant plates should have colonies - Kan (for me) should not have colonies
HOWEVER, for some reason, the Kan plate AND the Amp plate both displayed a lawn of bacterial colonies.
2) Grab 1 - 2 Falcon tubes. Fill each with 3 mL of LB and 3 microliters of Amp.
3) Obtain the minigrip sterile loops. Pick a colony on the plate (choose one).
4) Have a bunsen burner running. Run the sterile loop through the flame quickly and then gently touch a single colony.
5) Swirl the end of the loop in the Falcon tube. Dispose of the loop.
6) Repeat for second tube (pick a different colony)
7) place the tubes in the shaker in the other room (overnight).
October 13, 2005
I checked on the incubated petri lawns. Only one colony grew on the ligation plate. The (+) and (-) controls showed expected results - growth on the positive plasmid control - no growth on the negative water control.
I prepared another transformation from the previous ligation. Alain plated and incubated the transformation for me :)
--Yi-An 13:59, 13 October 2005 (EDT)
GROWING/PLATING THE COLONIES
October 14, 2005
I checked on the bacterial lawn of ligated DNA that i incubated overnight. There were a few colonies present across the streaked regions. (around 10 colonies).
So, apparently, I forgot (or just did not take into account) the possibility that the restriction cut DNA could re-ligate to form the original vector (promoter). This poses a problem because the re-ligated DNA would still be able to grow colonies on the bacterial petri lawn due to Amp resistance.
GROWING BACTERIA IN PREP FOR SECOND DIGESTION PROCEDURE:
1) Grab necessary materials:
- 10 Falcon tubes - LB solution - vial of Amp - innoculated bacterial lawn (from the other lab room) - Bunsen burner - toothpicks - Amp resistance petri plate
2) Label the Falcon tubes 1 thru 10 and initial each tube. Draw a grid of 16 onto amp resistant petri plate.
___|_1_|_2_|___
_3_|_4_|_5_|_6_
_7_|_8_|_9_|_10
___|___|___|___
3) Turn on the bunsen burner for sterilization purposes.
4) Pipet 3 mL of LB and 3 microliters of Amp into each Falcon tube.
5) Locate a bacteria colony on the petri lawn. take a toothpick and gently touch the center of the colony.
6) Patch (streak the bacterial colony using the flat end of the toothpick onto grid #1 of the petri plate). Drop the toothpick in Falcon tube #1. Cap the Falcon tube and set aside.
7) Repeat steps 5 thru 6 for 9 other colonies. Make sure the grid number that you streak a colony onto matches the tube that you drop the toothpick into. The petri grid will serve as the master plate for future reference.
8) Place the newly plated petri dish/grid into the incubator in the other lab room. Place the 10 Falcon tubes into the shaker overnight.
--Yi-An 13:59, 14 October 2005 (EDT)
PHOSPHOTASE DOUBLE DIGEST:
Another Alternative October 14, 2005
Alain explained that often when the Digest is only performed for a short period of time, it is highly probable that one of the restriction enzymes did not cut properly. Normally, when the digest runs overnight there is a lesser chance of this occurring - however it is still possible. When only one restriction enzyme cuts the DNA, the sticky ends are still present and since they are in close proximity, there is a higher chance of forming the original vector opposed to the desired vector with the DNA insert. The result of this will often be a high yield of bacterial colonies after the overnight incubation of the ligation/transformation DNA.
When checking the gels, it is hard to distinguish whether the DNA you have is that of the original vector or the desired one since the difference between the DNA lengths is very small.
When Digesting the DNA for short periods of time, you want to avoid this problem. Thus a double digest is performed.
After the DNA Digest is complete...
1) Pipet 1 microliter of phoshotase into ONLY the VECTOR (promoter) vial. DO NOT PLACE PHOSPHOTASE into the reporter vial or this will completely eliminate any chance for the desired product due to no possibility of liagation. The phosphtase will remove a phosphate from the 5' end of the singly-cut vector. Since the 3' end also does not possess a phosphate, there is no chance for the original singly cut site to re-ligate. The insert however, will have phosphates on both ends.
2) incubate the vial at 37 degree Celsius for 1 hour.
3) proceed with the gel electrophoresis, extraction, ligation, and transformation.
- Note: I will try to get a computer-drawn picture of this up asap.
--Yi-An 13:59, 14 October 2005 (EDT)
COLONY PCR PROTOCOL
Usually you should perform PCR once before growing/innoculating the bacteria (after transformation); however, since i wasn't aware of this i plated/gridded/transferred the bacterial colonies first. Thus, i had to prepare the PCR solution from the dilute DNA mixtures from the Falcon tubes.
1) Grab the 11 mini centrifuge tubes and label them from 1 - 11. The 11th eppendorf tube will serve as the negative control. Be sure to wear gloves. You do not want your DNA to contaminate the PCR tubes since this is a very sensitive procedure.
2) Obtain the PCR supermix from the refrigerator in the other lab room [it is in a red and white Invitrogen box that is surrounded by a clear drawstring mini-bag]. Obtain the primers VF2 and VR from the clear gridded box from the project 3 freezer. [VF2 is a primer that will anneal close to the EcoRI RE site and amplify in the forward direction, VR is a primer that will anneal close to the PstI RE site and will amplify in the reverse direction].
3) Take one of the mini eppendorf tubes. This will serve as your master mix tube [for a giant mix of everything needed for PCR minus the DNA]. Measure 120 microliters of supermix (after is has thawed) and add it to this mini eppendorf tube [this is for if you have 10 bacterial colonies]. To get the amount of supermix needed:
FUN ACTIVITY: Watch Perry do a "THAW DANCE" with the PCR Supermix :)
(number of bacterial colonies [or number of Falcon tubes prepped] + 2) X 10 = total amount of supermix needed
4) I added 1.8 microliters of each primer (VF2 and VR) to the master mix tube.
to obtain the amount of each primer needed:
(number of bacterial colonies [or number of Falcon tubes prepped] + 2) X 0.15 = amount of EACH primer needed
5) Pipet 10 microliters of master mix to each of the numbered mini-eppendorf tubes. the original master mix tube should have roughly 20 microliters of master mix remaining - this will serve as the negative control (DO NOT ADD DNA TO THIS TUBE).
EDIT: Alternate procedure
1) Grab the number of mini eppendorf tubes that you need.
2) Add 45 microliters of PCR Supermix to each tube
3) set up a separate mini eppendorf tube for the primer mixing. You will need 5 microliters of 2 micromolar VF2 and 5 microliters of 2 micromolar VR for each tube.
THus...
(5 microliters of VF + 5 microliters of VR) X number of pcr rxns you are doing = total volume of primer mix
4) perform a dilution of your template dna. 1:100. thus set up another set of eppendorf tubes and label them. Place 99 microliters of water in each tube as well as 1 microliter of the DNA into their respectively labeled tubes.
5) Add 10 microliters of primer mix to each of the tubes already containing supermix. Add 2 microliters of your template dilution to their respective tubes as well.
NOte: on the pcr machine - use "YK PCR" program to run.
to add loops press, "."
specifications:
- 10 minutes initial 95 degrees celsius
loop: - run at 95 degrees celsius for 1 min - run at 55 degrees celsius for 1 min - run at 72 degrees celsius for 1 min
final extention (extn) is 72 degrees celsius for 10 minutes final hold is 4 degrees celsius forever :)
press the inverted triangle once to save the program (do not hit twice otherwise it will delete the program) - followed by enter :)
When taking out the pcr tubes:
make sure to end the program and shut off the machine (switch is in the back) before openeing the top. If you don't do this, you will cause the block inside to form ice particles.
SETUP THE PCR MACHINE
Set the machine for: a 15 minute initial run at 95 degrees Celsius
The machine will cycle through 3 different temperatures.
95 degrees Celsius: The purpose of this high temperature is to denature the DNA and to cause the DNA strands to unwind and separate. During each cycle, the machine will have the tubes at this temperature for 30 seconds.
55 degrees Celsius: The purpose of this is to allow the primers to anneal to the separated strands of DNA. During each cycle, the machine will have the tubes at this temperature for 30 seconds.
68 - 72 degrees Celsius: The purpose of this to EXTEND the DNA (amplification). During each cycle, the machine will have the tubes at this temperature for 1.5 minutes. The longer the time interval for this EXTENSION, the longer the DNA amplification will be. This temperature is the optimal one for the attack polymerase to operate at.
After the PCR machine has completed 30 cycles (which should take roughly 2 hours), make a 1% Agarose gel and run the DNA from the PCR reaction with a 1 Kb Ladder (add loading dye to each DNA sample prior to loading into wells). If you are on a time crunch, you can leave it in the PCR machine because when it is done, it will automatically change the temperature to 4 degrees Celsius.
--Yi-An 13:59, 14 October 2005 (EDT)
October 14, 2005
friendly information. Must wait for the DNA to be miniprepped. There are 3 samples. I will sequence this tomorrow. The primers are located in the Project 3 refrigerator. The 3 DNA samples are located Q04121 (QPIlacI), J06800 (QPI241), J06801 (QPI265).
Note: elutions can achieve greater efficiency if they are eluted with hot water.
--Yi-An 13:59, 14 October 2005 (EDT)
October 15, 2005
-took the PCR tubes out of the machine and added 2 microliters of loading dye to each mini-eppendorf tube.
-prepared a 1% agarose gel and ran the gel
-prepared 2 liters of 1x TBE from 10X TBE.
Note: if the machine for double distilled water doesn't seem to work - it is probably hibernating [indicated by line (in the contour shape of a top hat]. If this is the case, follow the instructions on the sheet posted on the wall to the left of the back sink.
-took the gel to the imaging machine and got the following images: (will upload later tonight)
oops. so apparently i left the gel running for too long and thus i have to redo the PCR reaction and rerun the samples on 2.0% E-Gels.
HAPPY NEWS: i went to check on my innoculated bacteria grid lawn and found all 10 grid spaces had squiggles of red colonies :) assembly worked :)
PICTURE
PREPARING THE CULTURES FOR STORAGE
October 15, 2005
1) Obtain screw-capped tubes (2 for each colony/LB culture). I grabbed 20 screw-capped tubes and another 10 eppendorf tubes. Label the tubes for the # culture they correspond to (e.g. culture #1 should have two screw-capped tubes labeled #1 and initialized).
2) Turn on a bunsen burner for sterilization purposes. pipet 750 microliters of culture #1 into each of the respective tubes. Pipet 250 microliters of "50% glycerol in distilled water" into each of the tubes. You should have 1.5 mL of culture left in the tube. Repeat for the other 9 cultures.
3) Label the 10 eppendorf tubes 1 thru 10. Pipet/transfer the remaining 1.5 mL of each culture into their respectively labeled eppendorf tubes.
4) Cap the 10 eppendorf tubes and place them in the centrifuge for 1 minute at 13000 rpm.
5) Suction or simply dispose of the supernatant.
6) Grab 2 white storage boxes on the shelf. Label them with their respective contents - promoter/reporter used, date, name - whether its the glycerol mix or the centrifuged pellet.
7) Place the box with the glycerol mixes in the -80 degree Celsius freezer. Place the box of the centrifuged pellets in the project 3 student freezer.
LACI PROMOTER SEQUENCING PROTOCOL
October 15, 2005
Chris and I performed a bit of sequencing today :)
1) Obtain the 2 primers - VF2 and VR from the project 3 student refrigerator
2) Obtain the QPIs (in this case they were the constructs J06800 and J06801 - which were prepared by Andrew and Perry respectively).
3) Grab 2 empty eppendorf tubes and place 4 microliters of 20 micromolar primer VF into one eppendorf tube and 4 microliters of 20 micromolar primer VR. Place 54 microliters of double distilled water into each tube. This will create 2 micromolar primers.
4) Grab two PCR strips of tubes and two strip covers (these strips are located on the shelf of the left lab bench when you enter the lab room - they look like mini eppendorf tubes connected in sequence).
5) Pipet a construct (8 microliters) and a primer (4 microliters) into each tube. Specifically, for each construct you must have one tube with construct + VF2 and another tube with construct + VR. Thus the number of constructs X 2 = the number of tubes you will need. Make sure to take note of which constructs and which primers you use in each strip tube (make sure to match them with the name you assigned them in the placement of the sequencing purchase order).
6) Store any excess 2 microliter primer into the primer storage case found in the project 3 student refrigerator.
SEQUENCING PURCHASE ORDER
1) go to Genewiz.com
2) enter Alain's email - aviel@fas.harvard.edu
pw: seqforstu
3) choose the number of sequencing segments you want to order. choose "premix" (which indicates that you have already placed the primer in with the DNA). select "no" for overnight delivery.
3) On the next screen, choose DNA type: plasmid. Use the scroll down to choose this. Click fill to fill in the rest of the slots with the same.
4) label the sequencing segments with your initials then a three digit number like (e.g. YK001)
5) bp length. Go to the part.mit.edu site --> IGEM 2005 --> find the construct/QPI part --> find the length of the plasmid.
6) Get a purchasing number from Alain. Print out the sample information sheet and add it to the binder of sequencing orders.
Information for when the sequencing returns:
YK001 contains J06801 (construct) and VF2 (primer)
YK002 contains J06801 (construct) and VR (primer)
YK003 contains J06800 (construct) and VF2 (primer)
YK004 contains J06800 (construct) and VR (primer)
--Yi-An 13:59, 15 October 2005 (EDT)
October 18, 2005
- Grew cultures (innoculation protocol) for the QPIs and constructs using Katherine's frozen glycerols: 3 constructs: (AMP)
- pSBlAT3-1 - pSBlac3 - 1 - psBlAK3-1
3 QPIs: (KAN)
- QPI Q04520 pSB2K3 - QPI A04510 pSB2K3 - QPI Q04121 pSB2K3
For the QPIs, the protocol was a little different. Instead of adding 3 microliters of Amp as done with the constructs listed above, i added 2 microliters of KAN and 30 microliters of IPGE to 3 mL of LB. Later, i added the appropriate constructs/QPIs with sterile loops.
I finished sequencing the QO4121 QPI. Added 8 microliters of Q04121 and 4 microliters of primer VF2 (YK005) to one strip tube and 8 microliters of Q04121 and 4 microliters of primer VR to another tube (labeled YK006).
the order was placed for the last part to be sequenced.
Random readings:
Light and the Transcriptional Response of the Microcystin Biosynthesis Gene Cluster http://www.pubmedcentral.gov/articlerender.fcgi?tool=pmcentrez&artid=92160
Light induction of gene expression in Myxococcus xanthus http://www.pubmedcentral.gov/articlerender.fcgi?tool=pmcentrez&artid=304650
UV induction of coliphage 186 http://www.pubmedcentral.gov/articlerender.fcgi?tool=pmcentrez&artid=297649
October 19, 2005
- performed a miniprep on the QPIs and constructs that i innoculated last night - will probably run an E-gel to check the DNA lengths before proceeding to Restriction Digest
It's a lovely day :)
October 20, 2005
- performed a miniprep on the colonies 1, 2, 5, 6, 7, and 8.
- decided against running an e-gel and to continue with performing a pcr instead (revised the pcr protocol)
- started the assembly - innoculated R0051(AMP), QPI4510 (KAN), J06702 mCherry (AMP), J06800 (KAN).
- plan: tomorrow - miniprep the DNA and perform the digests; meeting about dna sequencing
October 21, 2005
- checked the sequences that were ordered by Alain (J06800 and J06801)
- ran the gel for the pcr reaction
- prepared the QPI, and my 6 colony minipreps for sequencing, gave the sequences to Alain for order placement
- ran the minipreps of the assembly pieces that i innoculated last night
- ran the digests for the assembly pieces (overnight)
October 22, 2005
- sequenced the six colonies from the first assembly (colonies 1, 2, 5, 6, 7, 8) and QPI Q04121
- made and ran a gel with the second assembly parts R0051, J06800, QPI Q04510, J06702 mCherry (pictures to come)
October 25, 2005
- extracted DNA from my frozen gels (J06702 and J06800), Perry extracted the DNA for R0051 (thanks!)
- Made and ran a gel for QPI Q04510 (since it did not show up as desired on the last gel) (pictures to come) - BLAH! the QPI DNA did not show up again. It just turned up as two long smudges on the gel. I will go back and check for the plasmid to ensure that no degradation occurred.
ANALYZING SEQUENCES
USE THE COMPUTER IN THE OTHER ROOM. IT IS THE ONE THAT IS RIGHT IN FRONT OF YOU WHEN YOU ENTER THE ROOM (BY THE WINDOWS). LOG IN.
1. Open the Genewiz account to view the sequences. Save them to the computer. Open the programs FINCHTV and VECTORNTIExplorer. Drag the sequence file into the screen of FINCH TV program.
2. Edit the BEGINNING AND THE END of the sequence by highlighting the sequence segment that does not have a clear, distinct sequence pattern. Highlight these bases (SHIFT + CLICK TO HIGHLIGHT) and change them to lower case (APPLE + "L"). Then Highlight and Copy the UPPER CASE SEQUENCE.
3. Click on "DNA/RNA" in the title bar. Scroll down and click "Create molecule." Fill out:
- General: Name of your Sequence (e.g YK001)
- DNA/RNA molecule: Linear
- sequence: Copy and Paste the UPPER CASE SEQUENCE here
4. Go to Parts.mit.edu and find the sequenced part you are looking to match (promoter) and click on biobricks in the scroll down --> Click "Features" and get sequence. Copy this sequence and save in the computer.
5. Find the 2 parts in the main database. Highlight them and click "Align molecules" If the sequences match, they will appear highlighted in yellow in the lower screen. Copy the highlighted segment.
6. Open VECTORNTI program. PASTE the COPIED sequences. The progam should show you where the sequence lies and where restriction sites lie.
... to be continued... there are some kinks in this protocol to be edited.
NOTE TO SELF: edit this when you perform the next sequencing.
October 26, 2005
- made/ran a gel with the QPI Q04510. Result: both the negative and positive trials ended up showing up as smears on the gel (picture to come)
- innoculated more QPI Q04510 to prep for another miniprep (to be performed tomorrow). I apparently used up all the DNA from the previous miniprep due to the number of gel trials i had.
Protocol
- found in "QIAGEN Plasmid Purification Handbook" under "Protocol: Plasmid or Cosmid DNA Purification Usin QIAGEN Plasmid Midi and Maxi Kits"
- check the incubator wrt austin's plate (next time i come in)
October 27, 2005
- performed a Midiprep of QPI Q04510
- followed the protocol in the QIAGEN Plasmid Purification Handbook under "Protocol: Plasmid or Cosmid DNA Purification Using QIGEN Plasmid Midi and Maxi Kits
October 28, 2005
- checked the DNA from the MIDIPREP by running an E-gel, strong band shown (picture to come)
- tried to sequence the YK-007 - YK020. However, I found that most of the sequences were garbled with "N"s and mutations. Thus, I'm going to redo the sequences. YK017 worked - there was a match for about 500 bps.
- digested Q04510. made and ran a gel of the midiprep. will hopefully run an extraction :)
October 29, 2005
- performed an extraction from the excised gels for QPI Q04510
- made 2 - 1 person gels
- used Christi's midiprep for R0051 and J06702 mCherry (thanks!) to perform a digest
- ran a gel of the digests and excised the DNA pieces (picture to come), performed extractions on the gel samples; strangely, the gel picture shows two bands for my promoter. Alain says that it could be due to incomplete digestion which would result in residual supercoiled dna.
November 1, 2005
morning:
- performed 9 extractions of DNA for J06702 mCherry and R0051 (due to previous complications)
- checked the extracted R0051 DNA against the original Midiprep DNA to see which segment contains the target DNA. To do this, I made and ran a gel to check lengths. The DNA is placed in the following order (from leftmost well to the right) - (picture to come)
1. 1 Kb Ladder
2. empty
3. R0051 Top Band
4. empty
5. R0051 Bottom Band
6. empty
7. R0051 Midiprep DNA
- performed another digest on R0051 (just in case the gel stuff is incorrect)
November 3, 2005
- made and ran a gel for the 2 R0051 Digest samples
- made TBE solution
- ran an e-gel to test the concentrated extraction DNA. The wells were set up left to right: 1 Kb ladder, R0051 top, and R0051 bottom (every other well - picture to come). I used 10 microliters of ladder, 10 microliters of DNA in each well (added with 3 microliters of loading dye). edit: The e-gel didn't show any signs of DNA, which seems to suggest that the DNA disappeared during extraction.... time for detective work :)
November 5, 2005
- performed a ligation and transformation on the samples.
- analyzed the e gel data and realized that the bands for all the past steps have been either too long or too short. One band of R0051 was located at 12000 bp (which seems to be an uncut plasmid) and the other resides at 2000 bp (which is half the length of the expected insert). This seems to indicate that either there was a mistake in either the digest or the original DNA sample.
- used the R0051 midiprep and performed another digest. Prepared 3 tubes. One tube was the normal digest with PstI and Spe1. The second tube contained everything but PstI. The third tube contained everything but SpeI. I placed the tubes into the water bath incubator overnight.
- plated the (+), (-), and Ligation plasmid for QPI Q04510 and J06702 mCherry ligation.
November 6, 2005
- made and ran a gel for the overnight digests. The Original Midiprep plasmid was also run in the gel as a control. Strange band patterns resulted. (picture to come)
- checked the transformation plates at 12:00 pm noon. no growth.
November 8, 2005
- checked my master plate that was incubated overnight. There was growth; however, the colonies were not red
- thus, i performed a miniprep on the inocculated bacteria.
- i performed a check-digest using XbaI and PstI to see if i could remove the reporter and check that it actually was inserted properly. Stored the miniprep materials in a new box. 12:20 pm - time of digest start; will return at 2:30 pm to remove it from the water bath.
- made and ran a gel for the digest check, the results seemed to indicate an uncleaved plasmid
November 9, 2005
- performed another check digest (suggested by Franklin). Performed a linearizing digest with EcoRI to check for the entire length of the DNA. Also performed a two RE digest - used EcoRI and PstI. Placed the stuff in the water bath incubator.
- took previous ligation and transformed it. Found out that the reason that my bacteria did not grow was because i plated it onto a KAN plate rather than an AMP plate. Since the vector is AMP resistant that determines was plate resistance to use.
- plated the transformation and incubated it
November 10, 2005
- made and ran a gel of the linear and double digested parts
November 13, 2005
- performed another transformation (could not find the earlier transformation) for the two paired assemblies R0051/J06702 mCherry; incubated the plates
November 15, 2005
- performed an innoculation and streaked the colonies onto a master plate
-the R0051/J06800 assembly yielded many colonies while the Q04510/J06702 mCherry Assembly only resulted in the growth of 4 colonies (not red)
November 16, 2005
- perform a miniprep on the innoculated bacteria
November 20-28, 2005
Thanksgiving break :) home sweet home :)
November 30, 2005
- ran two egels to check the two assembly halves
- the resulting gels indicated minute (if any) traces of DNA (John said it was just reflective light - boohoo)
- put all of the samples through the nanodrop - none of them displayed the appropriate peak at 260 nm. The concentrations are as follows:
R0051/J06800
Clone 1: 12.1 ng/microliter
Clone 2: 5.7 ng/microliter; 0.115 absorbance at 260 nm
Clone 3: 10.2 ng/microliter; 0.203 absorbance at 260 nm
Clone 4: 8.7 ng/microliter;0.174 absorbance at 260 nm
Clone 5: 16.1 ng/microliter; 0.322 absorbance at 260 nm
Clone 6: 10.3 ng/microliter; 0.206 absorbance at 260 nm
Clone 7: 9.2 ng/microliter; 0.184 absorbance at 260 nm
Clone 8: 8.3 ng/microliter; 0.166 absorbance at 260 nm
Clone 9: 9.0 ng/microliter; 0.179 absorbance at 260 nm
Clone 10: 6.3 ng/microliter; 0.127 absorbance at 260 nm
QPI Q04510/J06702 mCherry
Clone 1: 19.2 ng/microliter; 0.385 absorbance at 260 nm
Clone 2: 8.0 ng/microliter; 0.159 absorbance at 260 nm
Clone 3: 12.1 ng/microliter; 0.243 absorbance at 260 nm
Clone 4: 12.3 ng/microliter; 0/246 absorbance at 260 nm
December 2, 2005
- performed an innoculation of my R0051/Jo6702 mCherry - prepping for a MIDIPREP (innoculation placed in the incubator at 6:30 pm --> take it out 12/3 at 11:00 am or 12:00 pm)
- performed a PCR on all my other samples of the parallel assemblies
December 3, 2005
- made/ran gel for the PCR, PCR yielded bands for 3 of the Q04510 constructs but not for the R0051 constructs. The gels i ran have 5 microliters of the PCR DNA and 5 microliters of loading dye - pictures on the other computer
- centrifuged the innoculation for the MIDIPREP - froze the pellets because i discovered that an innoculation is necessary for the spectrum testing - not miniprep stuff
December 4, 2005
- performed an innoculation of my old constructs: R0051, J06702 mC, J06800, and QPI4510 (running the beginning in parallel to my pcr and first assembly checks.
- performed an innoculation of my first assembly to prep for miniprepping
- performed another innoculation of the very first prelim assembly with R0051/J06702 mC in order to test the spectrum of the reporter.
December 5, 2005
- performed minipreps on R0051, J06702 mC, J06800, and QPI4510; also performed minipreps on the R0051/J06800 assembly
- in my rush to fit time constraints since i had a class at 7:00 pm in the Quad - i accidentally miniprepped the R0051/J06702 mC constructs. Thus, i performed another innoculation of the very first prelim assembly with R0051/J06702 mC.
December 7, 2005
- note: i hate being sick... and it's too cold outside *boohoo*
- performed an e-gel on the first assembly and miniprep attempts- to no avail
- reinnoculated the bacteria for old constructs
- obtained nanodrop values for all the first assembly constructs - the values were too low for significant dna content
December 8, 2005
- performed a restriction digest, phosphotase digest, and made/ran a gel (first assembly check)
- performed a miniprep on all old constructs
December 9, 2005
- performed an innoculation of my old assembly: R0051/J06702
December 13, 2005
- perform an e-gel to check miniprep dna - Q04510 and J06800 appeared as clear bands
- perform nanodrop checks (2 microliters of DNA measured each time; water was used to blank the machine)
- J06702 mC: 90.9 ng/microliter; 1.818 at 260 nm (little peak) (when i attempted to concentrate the sample further - the microcon filter collapsed and fell out - thus i am performing another innoculation/miniprep to get sufficient DNA)
- J06800: 172.0 ng/microliter; 3.440 at 260 nm (peak)
- Q04510: 149 ng/microliter; 2.98 at 260 nm (peak)
- R0051: 60.4 ng/microliter; 1.208 at 260 nm (little peak); attempted to concentrate more: 107.0 ng/microliter; 2.141 at 260 nm
- R0051 (midiprep): 91.9 ng/microliters; 1.834 at 260 nm (peak)
- performed an innoculation of my old assembly: R0051/J06702 (3 - 3 mL innoculations)
- performed an innoculation of J06702 mC (5 samples of 10 mL LB innoculations using AMP)
