IGEM:The Citadel/Training: Difference between revisions
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=May Crash Course in Synthetic Biology= | |||
In order to get some serious training under our belt, do something that ''helps iGEM, and start work that can be credited towards our project'' we will be testing Biobrick promoter strength on May 28-June 4th. | In order to get some serious training under our belt, do something that ''helps iGEM, and start work that can be credited towards our project'' we will be testing Biobrick promoter strength on May 28-June 4th. | ||
Those performing the training, make sure that you are familiar with [http://www.jbioleng.org/content/3/1/4 "Measuring the activity of Biobrick Promoters Using an ''In vivo'' Reference Standard"] by ''Jason Kelly et al.''. The supplementary materials can be safely downloaded by clicking on[http://www.jbioleng.org/content/supplementary/1754-1611-3-4-s1.doc| this link here]. For a graphical summary of the protocol, travel over to the [http://partsregistry.org/Measurement/SPU/Measure Parts Registry Measurement Section]. | |||
* | * Note that we WILL NOT be doing Step #1 (synthesis and annealing of oligos to make a test promoter). Instead, we will be testing promoters that have been included with our distribution. | ||
=COMPLETE PROTOCOL= | |||
==[[Knight:Preparing_electrocompetent_cells| TK's PREPARING ELECTROCOMPETENT CELLS]]== | |||
===Materials=== | |||
==Materials= | |||
<u>Equipment</u> | |||
*-80°C freezer | *-80°C freezer | ||
*37°C incubator | *37°C incubator | ||
*Refrigerated centrifuge that accepts 225 mL culture tubes | *Refrigerated centrifuge that accepts 225 mL culture tubes | ||
<u>Chemicals and Reagents</u> | |||
*~500 mL LB Lennox supplemented with appropriate concentration of antibiotic if appropriate. | *~500 mL LB Lennox supplemented with appropriate concentration of antibiotic if appropriate. | ||
*~600 mL sterile deionized water chilled to 4°C | *~600 mL sterile deionized water chilled to 4°C | ||
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*Ice bucket and ice | *Ice bucket and ice | ||
*Dry ice, ethanol bath | *Dry ice, ethanol bath | ||
<u>Supplies</u> | |||
*Many 1.5 mL plastic tubes chilled to -80 °C | *Many 1.5 mL plastic tubes chilled to -80 °C | ||
*14 mL culture tube for starter culture | *14 mL culture tube for starter culture | ||
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*Pipets | *Pipets | ||
==Procedure== | ===Procedure=== | ||
#Prechill all tubes and pipets at 4°C or -80°C as appropriate. <br> Also rinse all flasks with H<sub>2</sub>O prior to autoclaving in order to remove residual detergents that may remain on glassware from dishwashing. This step may increase competency. Autoclaving with water, which is then discarded, is even better. | #Prechill all tubes and pipets at 4°C or -80°C as appropriate. <br> Also rinse all flasks with H<sub>2</sub>O prior to autoclaving in order to remove residual detergents that may remain on glassware from dishwashing. This step may increase competency. Autoclaving with water, which is then discarded, is even better. | ||
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# Store at -80°C | # Store at -80°C | ||
==Notes== | ===Notes=== | ||
*'''[[User:Rshetty|RS]] 17:49, 13 June 2006 (EDT)''': Dont' forget to use the conical bottoms on the swinging buckets in the centrifuge ... not the flat ones. Otherwise, your tubes will break. (Oops.) | *'''[[User:Rshetty|RS]] 17:49, 13 June 2006 (EDT)''': Dont' forget to use the conical bottoms on the swinging buckets in the centrifuge ... not the flat ones. Otherwise, your tubes will break. (Oops.) | ||
This protocol is for transforming plasmid DNA into ''Escherichia coli'' cells. | |||
====Materials==== | |||
===Materials=== | |||
*[[Electrocompetent Cells | Electrocompetent cells]] | *[[Electrocompetent Cells | Electrocompetent cells]] | ||
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*1mL [[SOC]] at room-temperature | *1mL [[SOC]] at room-temperature | ||
===Procedure=== | ====Procedure==== | ||
#Chill electroporation cuvettes, DNA samples and tubes on ice. | #Chill electroporation cuvettes, DNA samples and tubes on ice. | ||
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#If you don't have any transformants, plate the rest of the transformation in the morning. | #If you don't have any transformants, plate the rest of the transformation in the morning. | ||
==Notes== | ===Notes=== | ||
If you are in a hurry and your selection marker is ampicillin, you can go ahead and plate immediately because ampicillin takes a while to be pumped into cells at a high enough concentration to have an effect. | If you are in a hurry and your selection marker is ampicillin, you can go ahead and plate immediately because ampicillin takes a while to be pumped into cells at a high enough concentration to have an effect. | ||
[[ | ====[[Miniprep/Qiagen_kit| MINIPREP (BASED ON THE QIAGEN KIT)]]==== | ||
===Materials=== | |||
= | |||
==Materials= | |||
For purifying plasmid DNA from ''Escherichia coli'' cells, the [http://www1.qiagen.com/Products/Plasmid/QIAprepMiniprepSystem/QIAprepSpinMiniprepKit.aspx Qiagen Spin Miniprep Kit] produces quite reliable results. | For purifying plasmid DNA from ''Escherichia coli'' cells, the [http://www1.qiagen.com/Products/Plasmid/QIAprepMiniprepSystem/QIAprepSpinMiniprepKit.aspx Qiagen Spin Miniprep Kit] produces quite reliable results. | ||
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The LyseBlue indicator dye added to some of the buffers is Thymophthalein, pH shift from colorless to blue at pH 9.3 | The LyseBlue indicator dye added to some of the buffers is Thymophthalein, pH shift from colorless to blue at pH 9.3 | ||
==Protocol== | ===Protocol=== | ||
See [http://www1.qiagen.com/literature/handbooks/PDF/PlasmidDNAPurification/PLS_QP_Miniprep/1027678_HB_QP_0504_WW_LR.pdf here] or [http://www1.qiagen.com/literature/protocols/QIAprepMiniprep.aspx here] for the handbook for the Qiagen Spin Miniprep Kit. If you have never done this protocol before, read the the background information in the handbook (like the Important Notes section). It contains useful information. The following has been reproduced from the handbook and annotated based on experience with the kit. | See [http://www1.qiagen.com/literature/handbooks/PDF/PlasmidDNAPurification/PLS_QP_Miniprep/1027678_HB_QP_0504_WW_LR.pdf here] or [http://www1.qiagen.com/literature/protocols/QIAprepMiniprep.aspx here] for the handbook for the Qiagen Spin Miniprep Kit. If you have never done this protocol before, read the the background information in the handbook (like the Important Notes section). It contains useful information. The following has been reproduced from the handbook and annotated based on experience with the kit. | ||
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#Place the QIAprep column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50 μl Buffer EB (10 mM Tris·Cl, pH 8.5) or water to the center of each QIAprep spin column, let stand for 1 min, and centrifuge for 1 min.<br>''If you are concerned about the concentration of the DNA, you can alternatively add 30 μL water to the center of the column, incubate at room temperature on the bench for 5 mins and then centrifuge for 1 min. This will increase the concentration of DNA in your final sample which can be useful in some cases. See notes below for why you should elute in water rather than the Buffer EB they recommend if you plan to sequence your sample. Even if you are not sequencing, it may be beneficial to elute in water. For instance, if you elute in buffer EB and you are using this DNA in a restriction digest, then the additional salts in your sample can affect the salt content of your digest. This may matter with some finicky enzymes.'' | #Place the QIAprep column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50 μl Buffer EB (10 mM Tris·Cl, pH 8.5) or water to the center of each QIAprep spin column, let stand for 1 min, and centrifuge for 1 min.<br>''If you are concerned about the concentration of the DNA, you can alternatively add 30 μL water to the center of the column, incubate at room temperature on the bench for 5 mins and then centrifuge for 1 min. This will increase the concentration of DNA in your final sample which can be useful in some cases. See notes below for why you should elute in water rather than the Buffer EB they recommend if you plan to sequence your sample. Even if you are not sequencing, it may be beneficial to elute in water. For instance, if you elute in buffer EB and you are using this DNA in a restriction digest, then the additional salts in your sample can affect the salt content of your digest. This may matter with some finicky enzymes.'' | ||
==Notes== | ===Notes=== | ||
*If you are doing more than ~10 minipreps simultaneously, it can save time to switch to the vacuum manifold version of this protocol since you eliminate having to load and unload samples into the centrifuge. | *If you are doing more than ~10 minipreps simultaneously, it can save time to switch to the vacuum manifold version of this protocol since you eliminate having to load and unload samples into the centrifuge. | ||
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*Similarly, doing the elution in two steps (first a 30 μL elution and then a 20 μL dilution) can also slightly increase yields. | *Similarly, doing the elution in two steps (first a 30 μL elution and then a 20 μL dilution) can also slightly increase yields. | ||
== | ==[[Qiagen_Buffers| QIAGEN BUFFERS AND COLUMN RECYCLING]]== | ||
Clicking the link above will provide you with the compositions of the different buffers used by Qiagen, as well as instructions on how to conserve the column that are included with the kit. | |||
==[http://ginkgobioworks.com/support/ NEB/GINKGO'S 3A BIOBRICK ASSEMBLY MANUAL]== | |||
The above link redirects you to the protocol we will use for 3A standard assembly. | |||
==[[Knight:Restriction_Digest| TK's RESTRICTION DIGEST]]== | |||
TK's RESTRICTION DIGEST | |||
<u>Materials</u> | <u>Materials</u> | ||
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* 4°C forever (or until you pull the reaction out of the thermal cycler). | * 4°C forever (or until you pull the reaction out of the thermal cycler). | ||
7. Generally, use some method of DNA purification to eliminate enzymes and salt from the reaction. | 7. Generally, use some method of DNA purification to eliminate enzymes and salt from the reaction. | ||
==Also Worth Checking Out== | |||
[[Endy:Making_a_long_term_stock_of_bacteria| DREW ENDY'S PROTOCOL FOR PRODUCING A STOCK OF BACTERIA]] | |||
[[TOP10_chemically_competent_cells| OWW's TOP10 CHEMICALLY COMPETENT CELLS]] |
Latest revision as of 15:25, 28 May 2010
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May Crash Course in Synthetic BiologyIn order to get some serious training under our belt, do something that helps iGEM, and start work that can be credited towards our project we will be testing Biobrick promoter strength on May 28-June 4th.
COMPLETE PROTOCOLTK's PREPARING ELECTROCOMPETENT CELLSMaterialsEquipment
Chemicals and Reagents
Supplies
Procedure
Notes
This protocol is for transforming plasmid DNA into Escherichia coli cells. Materials
For the following, you need one per DNA sample
Procedure
NotesIf you are in a hurry and your selection marker is ampicillin, you can go ahead and plate immediately because ampicillin takes a while to be pumped into cells at a high enough concentration to have an effect. MINIPREP (BASED ON THE QIAGEN KIT)MaterialsFor purifying plasmid DNA from Escherichia coli cells, the Qiagen Spin Miniprep Kit produces quite reliable results.
Buffer P1
The buffer and RNaseA can also be ordered from Qiagen separately (catalog numbers 19051 and 19101). Buffer P2
Buffer P3 (not for spin columns, but for Qiatips, midi, maxi, giga kits)
Buffer N3
Buffer PB
Buffer PE
Buffer QBT equilibration buffer
Buffer QC wash buffer
Buffer QF elution buffer
Buffer QN
The LyseBlue indicator dye added to some of the buffers is Thymophthalein, pH shift from colorless to blue at pH 9.3 ProtocolSee here or here for the handbook for the Qiagen Spin Miniprep Kit. If you have never done this protocol before, read the the background information in the handbook (like the Important Notes section). It contains useful information. The following has been reproduced from the handbook and annotated based on experience with the kit. Protocol: QIAprep Spin Miniprep Kit Using a Microcentrifuge This protocol is designed for purification of up to 20 μg of high-copy plasmid DNA from 1–5 ml overnight cultures of E. coli in LB (Luria-Bertani) medium. For purification of low-copy plasmids and cosmids, large plasmids (>10 kb), and DNA prepared using other methods, refer to the recommendations on page 37. Please read “Important Notes” on pages 19–21 before starting. Note: All protocol steps should be carried out at room temperature. Procedure
Notes
QIAGEN BUFFERS AND COLUMN RECYCLINGClicking the link above will provide you with the compositions of the different buffers used by Qiagen, as well as instructions on how to conserve the column that are included with the kit. NEB/GINKGO'S 3A BIOBRICK ASSEMBLY MANUALThe above link redirects you to the protocol we will use for 3A standard assembly. TK's RESTRICTION DIGESTMaterials
Digest Mix Example - 50 μL reaction. 100 μL reactions are also common especially if your DNA to be cut is dilute.
Procedure
3. Add BSA to the tube.
4. Add appropriate amount of DNA to be cut to the tube.
5. Add 1 μL of each enzyme.
6. Place in thermal cycler (MJ Research, PT-200) and run digest protocol.
7. Generally, use some method of DNA purification to eliminate enzymes and salt from the reaction. Also Worth Checking Out |