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| - | {{:Team:DTU-Denmark/Templates/Standard_page_begin|Methods}}
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| - | == Project 1 ==
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| - |
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| - | === Week 1 ===
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| - | {|
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| - | |'''Day'''||'''What we did'''
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| - | |-
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| - | |Monday||Did something
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| - | |-
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| - | |Tuesday||Did something
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| - | |-
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| - | |Wednesday||Something
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| - | |-
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| - | |Thursday||
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| - | |-
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| - | |Friday||Something
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| - | |-
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| - | |Saturday||
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| - | |-
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| - | |Sunday||Did something
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| - | |}
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| - | === Week 2 ===
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| - | {|
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| - | |'''Day'''||'''What we did'''
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| - | |-
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| - | |Monday||Did something
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| - | |-
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| - | |Tuesday||Did something
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| - | |-
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| - | |Wednesday||Something
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| - | |-
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| - | |Thursday||
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| - | |-
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| - | |Friday||Something
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| - | |-
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| - | |Saturday||
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| - | |-
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| - | |Sunday||Did something
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| - | |}
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| - |
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| - | == Protocols ==
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| - |
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| - | === PCR protocol ===
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| - |
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| - | {| border="1"
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| - | | || Taq+PFU ($\mu$l) || Phusion ($\mu$l)
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| - | |-
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| - | | Enzyme|| align="right" | 0.5 || align="right" | 0.5
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| - | |-
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| - | | Forward primer (10 $\mu$l) || align="right" | 2.5 || align="right" | 5
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| - | |-
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| - | | Reverse primer (10 $\mu$l) || align="right" | 2.5 || align="right" | 5
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| - | |-
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| - | | dNTP (2 $\mu$l) || align="right" | 4 || align="right" | 4
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| - | |-
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| - | | DNA || align="right" | 1 || align="right" | 1
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| - | |-
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| - | | Buffer 10x || align="right" | 10 || align="right" | 10
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| - | |-
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| - | | Water || align="right" | 79.5 || align="right" | 74.5
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| - | |-
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| - | |align="right" | Total || align="right" | 100 || align="right" | 100
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| - | |}
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| - |
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| - |
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| - | === PCR program design ===
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| - | # Initial denaturation for 2 minutes at 95⁰C.
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| - | # Denature for 1 minute at 95⁰C.
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| - | # Anneal primers for 30 seconds at temperature ~5⁰C below melting temperature of primers.
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| - | # Extend DNA at 72⁰C using each 1-2 minutes per kilobase of product, depending on polymerase used (see manufacturer’s instructions).
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| - | # Repeat steps 2-4 for 25-30 cycles.
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| - | # Final extension for 10 min at 72⁰C
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| - |
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| - |
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| - | === PCR product purification using NucleoSpin ===
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| - | # Mix 1 volume of sample with 2 volumes of NT buffer in an 1,5 ml Eppendorf tube.
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| - | # Place a column into a 2 ml collection tube and load the sample.
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| - | # Centrifuge at 11.000 g for 1 min.
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| - | # Discard flow through and place the column back into the collection tube.
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| - | # Add 600 µl NT3 buffer and centrifuge at 11.000 g for 1 min.
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| - | # Discard flow through and place the column back into the collection tube.
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| - | # Centrifuge at 11.000 g for 2 min to remove NT3 buffer. Discard flow through.
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| - | # Place the column into a clean 1,5 ml Eppendorf tube.
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| - | # Add 30 µl of water or NE buffer and incubate for 1 min to increase the yield of eluted DNA.
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| - | # Centrifuge at 11.000 g for 1 min.
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| - |
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| - | === Plasmid puriification ===
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| - | This purification is based on the “Zyppy Plasmid Miniprep Kit”
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| - |
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| - | Amounts of bacterial culture: According to Zyppy the purification can be done on 600 ul cell culture, but our experience suggests that it is not enough for further processing/use of the DNA. We use 2-4 ml of cell culture.
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| - |
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| - | # Initial steps:
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| - | #* Add 1.5 ml of cell culture in LB medium to 2 ml eppendorf tube.
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| - | #* Spin at 15.000 g for 2 min.
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| - | #* Discard supernatant
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| - | #* Add 1.5 of cell culture (for a total of 3 ml cell culture)
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| - | #* Spin at 15.000 g for 2 min.
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| - | #* Remove as much supernatant as possible – pipette carefully. This is (the only) point of no return! To stop, freeze the pellet.
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| - | #* Add 600 ul of TE-buffer. Ensure that the pellet is completely suspended.
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| - | # Add 100 ul 7x lysis buffer. Remember not to process more than 10 minipreps at a time.
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| - | # Add 350 ul cold neutralization buffer. Mix gently and thoroughly (= all the way through ≠ violently)!
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| - | # Spin at 15.000 g for 5 min.
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| - | # Transfer the supernatant to the columns; be careful not to get some of the pellet! It’s better to leave some supernatant than to get some of the pellet. Several “lysis” can be poured together to up-concentrate.
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| - | # Spin at 15.000 g for 30 sec.
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| - | # Discard flow-through.
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| - | # Add 200 ul endo-wash-buffer
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| - | #* Spin at 15.000 g for 30 sec.
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| - | # Add 400 ul zyppy wash buffer
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| - | #* Spin at 15.000 g for 30 sec.
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| - | # Transfer columns to clean 1.5 ml eppendorf tubes. Be careful when removing the tubes, the buffer may not touch the tip of the column! (if it happens, spin again).
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| - | # Elute DNA in 30-100 ul of buffer of choice (TE/H2O/restriction buffer/Zyppy elution buffer). Add the buffer to the center of the column, but without touching the column material! If H2O, wait 5 min before proceeding to the final centrifugation step, as DNA is not easily suspended in water.
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| - | # Spin at 15.000 g for 30 sec.
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| - | # Check the purification by running a gel (at least until we get experienced with a high success rate).
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| - |
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| - |
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| - | === <Protocol-name-5> ===
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| - | Text..
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| - |
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| - | === <Protocol-name-6> ===
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| - | Text..
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| - |
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| - | === <Protocol-name-7> ===
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| - | Text..
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| - |
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| - |
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| - | {{:Team:DTU-Denmark/Templates/Standard_page_end}}
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