MIT Synbio:IAP

http://farm5.static.flickr.com/4090/5039587233_186da9193f_z.jpg From the Cambridge 2010 iGEM team

Overview
2 Intro classes: 1 day with two info sessions for people to come and see if they want to take the class or not Class 0: for people who haven't taken 7.012: DNA What is synthetic biology What are biobricks circuit concepts, circuit design (and/ or gates, NOR gates, etc.)/ feedback iGEM What they're doing: bioluminescence Techniques to teach: 1. Digestion: 1.5 hr		2. Ligation: 1.5 hr, do at the end 3. Gel extract + nanodrop: 20 min prep, 30 min run; 10 min cut, 20 min spin, 5 min nanodrop: 1.5 hr		4. Transformation: 1 hr, 1 hr grow, 20 min plating 5. Picking colonies 20 min--> cell stock--> grow up (us) 6. Miniprep: 1.5 hr, nanodrop 5 min 7. Restriction map
 * Take place before EHS training
 * things that have been done in the past
 * potential projects
 * Circuits
 * what you can do.
 * registry
 * Lab tour: pipetting, sterile
 * (Minipreps)
 * We need to make plates, gels
 * Labeling

Restriction Digest
Recall from your lecture on Biobrick assembly that the cut sites EX-SP surrounding the desired gene allow for restriction enzymes to create sticky ends that can then be used to ligate parts together. In this lab, we will be using the restriction enzymes XbaI, SpeI, and PstI to cut our plasmid DNA.


 * Receive 2 tubes of DNA from group leaders. One contains your promoter plasmid, and the other your protein plasmid. They each contain 15uL of DNA in solution.
 * You will do all calculations before you begin digestions. First, find the concentration of your DNA. Each tube is different. Then, knowing the concentration of your DNA is in ug/mL, calculate the ug of DNA in your tube.
 * You will use 10 units of enzyme per ug of DNA in your tube. Use the table below to calculate how many uL of enzyme you will use to digest your DNA.

Antarctic Phosphatase Treatment of Backbone
h3. Treatment Protocol

Antarctic Phosphatase treatment is done to phosphatase the backbone used for plasmid ligations. This is done to reduce the self ligation of a backbone digested with enzyme(s) creating compatible sticky ends and hence enhance the S/N ratio of transformations.


 * 1) Use 5 units of Antarctic phosphatase per 1µg of DNA (over digesting by factor of  X)
 * 2) Calculate volumes
 * 3) * DNA µg = amount of DNA in backbone digest = X
 * 4) * Enzyme volume = X/5 uL (minimum of 1 uL)
 * 5) * Buffer is dilution factor x dilution of the total volume.
 * 6) Order of filling
 * 7) * Backbone Digestion
 * 8) * Antarctic Phophatase Buffer
 * 9) * Antarctic Phosphatase
 * 10) Incubate for 15 minutes at the specified temperature for the enzyme (37C).
 * 11) Heat Inactivate for 5 minutes at 65C.
 * 12) Keep the buffer on ice and the antarctic phosphatase in the benchtop coolers when on the bench.

h3. CIP Treatment Setup

Enzyme: Antarctic Phosphatase Reference: |http://www.neb.com/nebecomm/products/productM0289.asp Concentration: 5 U/uL Volume of enzyme required = Amount of DNA digested / 5 uL (Minimum 1 uL)


 * Component || Volume ||
 * Backbone Digestion | Y uL |
 * Antarctic Phosphatase | Z uL |
 * NEB Antarctic Phosphatase Buffer | (Y+Z)/9 uL |

h3. Cycle Setup


 * 1) 37C for 15 minutes.
 * 2) 65C for 5 minutes.
 * 3) Hold at 10C.

Gel Extraction
h3. Preparing the Gel


 * 1) Dissolve UltraPure agarose to a final concentration of 1% in TAE buffer in a glass bottle.
 * 2) Heat the solution in the microwave with frequent stirring to dissolve the agarose homogenously.
 * 3) Place the solution in a 55C water bath for 15 mins.
 * 4) Add 2 µl SYBR-safe per 50 ml of the solution and mix well.
 * 5) Pour 50ml of solution per small gel tray. (the gel trays and combs should be pre-cleaned with water and wiped dry).
 * 6) * Note for combs: 15-well combs hold about 6 ul liquid, 12-well combs hold about 15 ul, 8-well combs hold about 20 ul
 * 7) ** Taping two 8-well comb wells together results in a well that holds up to 100 ul
 * 8) ** Taping three 8-well comb wells together result in a well that holds up to 200 ul
 * 9) Use 120ml per large gel tray.
 * 10) Wait for the gels to solidify.
 * 11) Label and store at 4C.

h3. Running the Gel

When doing gel extraction, it is important to run both an analytical gel (to view under UV) and an extraction gel (from which bands are excised). UV damages DNA, and so we dont want to expose our extracted DNA.

Analytical Gel:

The analytical gel should have between 20 and 100 ng of DNA in each well. It should be an exact copy of the extraction gel with respect to position, voltage, and run time.

Extraction Gel:

This should be the rest of the digestion(s).

The analytical and extraction gels can technically be part of the same physical gel. Make sure to separate with a razor blade before imaging.

Refer to Gel Prep protocol above to determine the amounts of liquid to load for the specific well.

Appropriate Hyperladder to be used for PCR product which is linear. Usually Hyperladder I will be used.


 * 1) While casting gel, add two sets of lanes; use one set to load an analytical gel.
 * 2) Add gel loading buffer (Orange G 6X), add 1X to 5X of DNA (it helps DNA sink into the bottom of the well) to DNA.
 * 3) Make sure there is enough 1xTAE in the plate holder.
 * 4) Load 5ul of appropriate hyperladder to one of the lanes.
 * 5) Load appropriate amount of DNA (mixed with the buffer) in each well.
 * 6) Set the timer and voltage to 120V and 60 min.

h3. Analytical Gel Annotation

The following things need to be added to the analytical gel image BEFORE it is posted to the wiki:
 * Label each lane with part number and amount of DNA loaded
 * Label each band with length and proposed identification
 * Include wt% agarose, run time, and voltage

h3. Gel Extraction Protocol using Zymo kit (preferred if available)


 * 1) Place the extraction gel on the blue light table.
 * 2) Cut out the appropriate bands. Place into 2mL microtube(s).  Try to cut out as small a piece as possible while still getting all the DNA.
 * 3) Weigh gel slice (tare with empty microtube). Add 3 volumes of ADB buffer per mg of gel (so a 100mg gel gets 300 uL of ADB buffer).
 * 4) Incubate at 55C for 10 minutes. Make sure that the gel is completely dissolved.
 * 5) Add dissolved gel solution to Zymo column in collection tube. Max volume is 800 uL at a time.
 * 6) Spin 14000 rpm for 30 sec.
 * 7) Discard liquid in collection tube.
 * 8) Repeat step 5-7 if had more than 800 uL dissolve gel.
 * 9) Add 200 uL DNA wash buffer.
 * 10) Spin 14000 rpm 30 seconds.
 * 11) Discard liquid in collection tube.
 * 12) Add 200 uL DNA wash buffer
 * 13) Spin 14000 rpm 1 min.
 * 14) Discard liquid in collection tube.
 * 15) Spin 14000 rpm 1 min one more time (dry spin).
 * 16) Discard collection tube (but not the column).
 * 17) (Optional: 2nd dry spin into clean collection tube.)
 * 18) Place column in a clean labeled microtube.
 * 19) Add 10 uL (min 6 uL for higher DNA concentration) of sterile DDH2O to top of column. Water should be pipetted directly onto center of filter.
 * 20) Incubate at RT 1 min (or longer).
 * 21) Spin 1 min at 14000 rpm.  Discard the column.
 * 22) Measure the concentration on the nanodrop.  (You may recover the 1uL from the nanodrop if needed.)

h3. Gel Extraction Protocol using QIAquick Gel Extraction Kit:


 * 1) Cut the gel to separate analytical and extraction gel; place analytical gel in UV illuminator.
 * 2) Look at the gel under low wavelength UV (high wavelengths will denature DNA). Quickly take a polaroid image and shut OFF the UV.
 * 3) Cut extraction gel under white light; avoid UV illuminating the extraction gel as this drastically decreases the DNA yield. If necessary, stain with Methyl Blue.
 * 4) Place the cut bands in 2ml Eppendorf tubes; Weigh slices; No more than 400mg per tube
 * 5) Add 3 volumes of Buffer QG to 1 volume of gel (100mg ~ 100ul)
 * 6) Incubate at 50C for 10min or until gel is dissolved; vortex every 2-3 min
 * 7) Confirm that color of mixture is yellow (if not, add 10ul of 3M NaAc, pH 5.0)
 * 8) Add 1 gel volume of isopropanol
 * 9) Add max of 770ul to QIAquick column and centrifuge for 1 min (max speed, \~13,000rpm, RT)
 * 10) Discard flow-through and place column back in tube.
 * 11) If needed, add rest of mixture to same tube (up to additional 770ul), spin, and discard flow-through
 * 12) Wash: add 0.75ml Buffer PE(make sure that the buffer has ethanol added to it) to column and centrifuge for 1 min
 * 13) Discard flow-through & centrifuge for 1 min
 * 14) Place column into clean Eppendorf tube
 * 15) Add 50ul Buffer EB or water to center of membrane
 * 16) Let stand at RT for 3 min
 * 17) Centrifuge for 5 min
 * 18) Measure the concentration using the UV spectrophotometer.

Ligation Protocol

 * Enzyme: T4 DNA ligase
 * Enzyme reference: |http://www.neb.com/nebecomm/products/productM0202.asp


 * 1) Enter the names of the plasmids from which the backbone and insert is obtained in the [Excel worksheet|https://wikis.mit.edu/confluence/download/attachments/63930612/ligation+setup.xls] or the [wiki version|Ligation Calculator]. Also enter the lengths of the vector and insert fragments. This can be obtained by looking at the components section of the menu on the left hand side of the relevant plasmid display in Vector NTI.
 * 2) The spreadsheet fills out the other columns to do a ligation with 100 ng of the vector fragment and a 3:1 molar ratio of vector to insert DNA.
 * 3) All ligations are typically done at RT for 2 hours, however difficult ligations or ligations with low concentration of vector or inserts (<10 ng/µl) can be done for 16 hours at 16C.
 * 4) Please keep the buffer on ice at all times when out of \-20C. Keep the enzyme in the benchtop coolers at all times when out of the \-20C

Transformations
Can leave the cells in the incubator for up to 18 hours but no more.
 * 1) Make sure that the incubator (30/37C) and water bath (42C) are ON
 * 2) Make sure required antibiotic plates are present. Check the antibiotic resistance on the plasmid map in Vector NTI.
 * 3) Take the DNA out of \--20 frig, let it thaw
 * 4) Make sure that all of the required reagents/DNA etc are present at the site of transformation before you take the cells out of the \-80.
 * 5) Thaw the competent cells on ice for 7-8 min.
 * 6) Add 1.0 µl of DNA (about 10ng) into the liquid (Don’t vortex). Tap the sides of the tube to mix. If transforming multiple plasmids, add 10 ng of each.
 * 7) Incubate the cells on ice for 30 min
 * 8) Heat shock the cells for EXACTLY 30 sec at 42 C water bath.
 * 9) Place on ice for 2 min.
 * 10) Add 0.9ml of room temperature S.O.C medium to each tube (S.O.C is made by dissolving 0.5 ml of 20% glucose in 25 ml of SOB. Make sure that the SOC is clear and not cloudy/ contaminated.)
 * 11) Shake the tubes at 37 C, 280 rpm for 60 min or 30 C for 90 min
 * 12) Plate 20uL on one side of a plate
 * 13) Spin remaining cells at 5000rpm for 1 min, pour off supernatant, resuspend cells in remaining liquid (DO NOT vortex) and plate on other side of plate.
 * 14) Incubate plates upright for 20 min., then upside down overnight (12-14 h) at 37 C or 16-18h at 30C.

Making Cell Stocks
h3. Instructions


 * 1) Acquire 500 uL of freshly grown cells in liquid culture. This  usually comes from the leftover culture that was grown for the miniprep.  Fresh Culture can also be grown by inoculating a single colony in TB  with appropriate antibiotic marker.
 * 2) Add 500 uL of culture to microtube.
 * 3) Add 500 uL of filter sterilized 30% v/v glycerol in water to  culture.
 * 4) Label the tube\!
 * 5) Place in appropriate box in \-80C freezer.

Culture Inoculation

 * 1) Decide on number of tries to be setup accordingly. 6 is a good number to try the first time.
 * 2) Aliquot X ml (X = Y number of tries * 3.5, where Y = number of tries.) of TB into a clean (autoclaved) beaker or flask. LB can be used if TB is not available.
 * 3) Add appropriate antibiotic at a 1:1000 dilution to the above solution. The antibiotic should correspond to the plasmid's antibiotic resistance. Since we will be using 1000x stocks of antibiotic, one would add 2 uL of antibiotic for 2 mL of total solution, 2.5 for 2.5 mL, etc.
 * 4) Take clean and autoclaved 14 ml polystyrene tubes (Called "Falcon tubes") and place them on a rack. Label them clearly. Aliquot 3.5 ml of TB/LB into each 14 ml tube.
 * 5) Use a sterile wooden applicator (autoclaved) or pipette tip to carefully pick an individual colony and dip the colony end of the applicator / tip into a 14 ml tube. Repeat this Y times using a fresh applicator each time. Make sure to pick a single colony per try.
 * 6) Place the tubes in the 37C shaker at 280-300 rpm and grow the cells for 12-14 hours. The tubes should be murky after the overnight growth.

Miniprep Protocol

 * 1) Make sure provided RNase A solution has been added to Buffer P1 before use. One vial of RNase A per bottle of Buffer P1 to give final concentration of 100ug/mL. If you're the one adding, initial top and check box on cap\!
 * 2) Buffer P1 will be in the fridge.
 * 3) Add ethanol to Buffer PE before use and then check mark on cap.
 * 4) Check Buffers P2 and N3 for precipitates, if any redissolve by placing in water bath at 37C Do NOT vortex.
 * 5) Add the provided LyseBlue reagent to Buffer P1 and mix before use. Use one vial LyseBlue per bottle of P1 to achieve 1:1000 dilution. If you're the one adding, initial top and check the box on cap.

h3. Steps


 * 1) Transfer 1-5mL of overnight culture of plasmid cells into 2ml microcentrifuge collection tubes (1 per try) provided in the kit. Pellet for 1 min. Decant all the liquid and add 1 ml of the culture into the corresponding tube. Make sure not to mix up the tries.
 * 2) Resuspend pelleted cells in 250 uL Buffer P1 and transfer to microcentrifuge tube.
 * 3) Add 250uL Buffer P2 and mix thoroughly by inverting tube 4-6 times. Do NOT vortex. Mixture turns blue.
 * 4) Add 350uL of Buffer N3 and mix IMMEDIATELY by inverting tube 4-6 times. Do NOT vortex. Mixture is no longer blue.
 * 5) Centrifuge 10min at 13,000 rpm in table-top centrifuge.
 * 6) Apply the supernatant to a QIAprep spin column by decanting. Do NOT get any of the sticky precipitate.
 * 7) Centrifuge for 30 - 60s at 13000rpm. Discard flowthrough.
 * 8) Wash the QIAprep column by adding 0.5 mL Buffer PB.
 * 9) Centrifuge for 30 - 60s at 13000rpm. Discard flowthrough.
 * 10) Wash the QIAprep column by adding 0.75 mL Buffer PE.
 * 11) Centrifuge for 30 - 60s at 13000rpm. Discard flowthrough.
 * 12) Place the QIAprep column in a clean 1.5mL microcentrifuge tube. To elute DNA, add 50uL Buffer EB to center of each column. Be careful NOT to pierce column.
 * 13) Let stand for 1 minute.
 * 14) Centrifuge for 60s at 13000rpm.
 * 15) Remove column and discard, tube now contains DNA.
 * 16) Go to NanoDrop and spec DNA.

PCR
h3. Assembling Reaction


 * 1) Get PCR tube (0.6mL tubes)
 * 2) Thaw Pfx supermix on ice
 * 3) Get primers for gene of interest


 * 1) Add the following:


 * Volume| Item|
 * 22.5uL| Pfx Supermix|
 * 1uL| 5uM Forward Primer|
 * 1uL| 5uM Reverse Primers|
 * 0.5uL| Template DNA|

h3. Calculating reaction conditions


 * 1) Use idtdna.com or VectorNTI to calculate primer temperatures of primers without common overhangs (Base pairs 30 to end when read 5' to 3')


 * Primer|Melting Temp|
 * FW| |
 * RV| |


 * 1) Pfx elongates at a rate of 500bp per 30s. Look up the length of the gene of interest and calculate time of elongation.

h3. Programming a thermocycler

..... need to look up what kind we have in TK's lab

A typical cycle follows:

Initial Denaturation: 5min @ 95C

Loop (30-35 cycles) Denaturation: 30s @ 95C Annealing: 30s @ temperature calculated above (55-65C normally) Elongation: 30s per every 500 base pairs @ 68C

Final Elongation: 15min at 68C Store: 16C

Restriction Mapping

 * 1) Choose a restriction enzyme such that the enzyme(s) cut both the vector and the insert and the bands can be distinguished reasonably on a gel to identify the correct try. Vector NTI should be used to design restriction maps.
 * 2) Make sure to create a gel map on vector NTI for the restriction digest.
 * 3) Restriction maps should be set up just like digestions are set up. Carefully calculate the amount of DNA you would need to digest. You need at least 50ng of DNA per band to see it on the gel. The DNA for each band is proportional to its size : for example if you expect to see a 500 bp band and a 4.5 kb out of a 5 kb plasmid after your digest, you need to digest at least 0.5 µg as they will be divided as 450ng of the 4.5kb band and 50ng of the 500 bp band.
 * 4) Keep the total volume as low as possible.
 * 5) Make gels as per gel extraction protocol.
 * 6) Run on the agarose gel for as long as required to obtain maximum resolution.
 * 7) Compare with the expected pattern of bands and pick out the correct try/tries for maxiprep transformation.

Sequencing
Template Total Mass (Read from table) / Concentration of Template (Side of tube) = X Repeat this table for each tube with the different primers. Remember, each tube has only ONE primer
 * 1) Ask an instructor to help fill out a sequencing form with genewiz on the iGEM account.
 * 2) Go to this link and find the recommended amount of DNA needed for the plasmid. Might need to lookup plasmid size in VectorNTI map.
 * 3) Read the concentration off the side of template DNA and use the following formula:
 * 1) Obtain a strip PCR tube and cut to length of sequencing samples
 * 2) For each sequencing primer, assemble the following in each tube:
 * Volume | Item |
 * X uL | Template DNA |
 * (10-X)uL | sterile H2O |
 * 1) To each tube being submitted (number of seq primers being used, add:
 * Volume | Item |
 * 5uL | 5uM a single sequencing primer |
 * 1) Tubes and printout of order confirmation are put into a Ziploc bag and dropped into submission box in Building 68, first floor. Time cut-off each day for sequencing is now 3pm.