Team:DTU-Denmark/Technical stuff lab
From 2011.igem.org
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== Protocols == | == Protocols == | ||
- | === | + | === PCR protocol === |
- | + | ||
- | === | + | {| border="1" |
- | + | | || Taq+PFU ($\mu$l) || Phusion ($\mu$l) | |
+ | |- | ||
+ | | Enzyme|| align="right" | 0.5 || align="right" | 0.5 | ||
+ | |- | ||
+ | | Forward primer (10 $\mu$l) || align="right" | 2.5 || align="right" | 5 | ||
+ | |- | ||
+ | | Reverse primer (10 $\mu$l) || align="right" | 2.5 || align="right" | 5 | ||
+ | |- | ||
+ | | dNTP (2 $\mu$l) || align="right" | 4 || align="right" | 4 | ||
+ | |- | ||
+ | | DNA || align="right" | 1 || align="right" | 1 | ||
+ | |- | ||
+ | | Buffer 10x || align="right" | 10 || align="right" | 10 | ||
+ | |- | ||
+ | | Water || align="right" | 79.5 || align="right" | 74.5 | ||
+ | |- | ||
+ | |align="right" | Total || align="right" | 100 || align="right" | 100 | ||
+ | |} | ||
- | |||
- | |||
- | === | + | === PCR program design === |
- | + | # Initial denaturation for 2 minutes at 95⁰C. | |
+ | # Denature for 1 minute at 95⁰C. | ||
+ | # Anneal primers for 30 seconds at temperature ~5⁰C below melting temperature of primers. | ||
+ | # Extend DNA at 72⁰C using each 1-2 minutes per kilobase of product, depending on polymerase used (see manufacturer’s instructions). | ||
+ | # Repeat steps 2-4 for 25-30 cycles. | ||
+ | # Final extension for 10 min at 72⁰C | ||
+ | |||
+ | |||
+ | === PCR product purification using NucleoSpin === | ||
+ | # Mix 1 volume of sample with 2 volumes of NT buffer in an 1,5 ml Eppendorf tube. | ||
+ | # Place a column into a 2 ml collection tube and load the sample. | ||
+ | # Centrifuge at 11.000 g for 1 min. | ||
+ | # Discard flow through and place the column back into the collection tube. | ||
+ | # Add 600 µl NT3 buffer and centrifuge at 11.000 g for 1 min. | ||
+ | # Discard flow through and place the column back into the collection tube. | ||
+ | # Centrifuge at 11.000 g for 2 min to remove NT3 buffer. Discard flow through. | ||
+ | # Place the column into a clean 1,5 ml Eppendorf tube. | ||
+ | # Add 30 µl of water or NE buffer and incubate for 1 min to increase the yield of eluted DNA. | ||
+ | # Centrifuge at 11.000 g for 1 min. | ||
+ | |||
+ | === Plasmid puriification === | ||
+ | This purification is based on the “Zyppy Plasmid Miniprep Kit” | ||
+ | |||
+ | 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. | ||
+ | |||
+ | # Initial steps: | ||
+ | #* Add 1.5 ml of cell culture in LB medium to 2 ml eppendorf tube. | ||
+ | #* Spin at 15.000 g for 2 min. | ||
+ | #* Discard supernatant | ||
+ | #* Add 1.5 of cell culture (for a total of 3 ml cell culture) | ||
+ | #* Spin at 15.000 g for 2 min. | ||
+ | #* Remove as much supernatant as possible – pipette carefully. This is (the only) point of no return! To stop, freeze the pellet. | ||
+ | #* Add 600 ul of TE-buffer. Ensure that the pellet is completely suspended. | ||
+ | # Add 100 ul 7x lysis buffer. Remember not to process more than 10 minipreps at a time. | ||
+ | # Add 350 ul cold neutralization buffer. Mix gently and thoroughly (= all the way through ≠ violently)! | ||
+ | # Spin at 15.000 g for 5 min. | ||
+ | # 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. | ||
+ | # Spin at 15.000 g for 30 sec. | ||
+ | # Discard flow-through. | ||
+ | # Add 200 ul endo-wash-buffer | ||
+ | #* Spin at 15.000 g for 30 sec. | ||
+ | # Add 400 ul zyppy wash buffer | ||
+ | #* Spin at 15.000 g for 30 sec. | ||
+ | # 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). | ||
+ | # 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. | ||
+ | # Spin at 15.000 g for 30 sec. | ||
+ | # Check the purification by running a gel (at least until we get experienced with a high success rate). | ||
+ | |||
=== <Protocol-name-5> === | === <Protocol-name-5> === |
Revision as of 21:28, 19 September 2011
Methods
Contents |
Project 1
Week 1
Day | What we did |
Monday | Did something |
Tuesday | Did something |
Wednesday | Something |
Thursday | |
Friday | Something |
Saturday | |
Sunday | Did something |
Week 2
Day | What we did |
Monday | Did something |
Tuesday | Did something |
Wednesday | Something |
Thursday | |
Friday | Something |
Saturday | |
Sunday | Did something |
Protocols
PCR protocol
Taq+PFU ($\mu$l) | Phusion ($\mu$l) | |
Enzyme | 0.5 | 0.5 |
Forward primer (10 $\mu$l) | 2.5 | 5 |
Reverse primer (10 $\mu$l) | 2.5 | 5 |
dNTP (2 $\mu$l) | 4 | 4 |
DNA | 1 | 1 |
Buffer 10x | 10 | 10 |
Water | 79.5 | 74.5 |
Total | 100 | 100 |
PCR program design
- Initial denaturation for 2 minutes at 95⁰C.
- Denature for 1 minute at 95⁰C.
- Anneal primers for 30 seconds at temperature ~5⁰C below melting temperature of primers.
- Extend DNA at 72⁰C using each 1-2 minutes per kilobase of product, depending on polymerase used (see manufacturer’s instructions).
- Repeat steps 2-4 for 25-30 cycles.
- Final extension for 10 min at 72⁰C
PCR product purification using NucleoSpin
- Mix 1 volume of sample with 2 volumes of NT buffer in an 1,5 ml Eppendorf tube.
- Place a column into a 2 ml collection tube and load the sample.
- Centrifuge at 11.000 g for 1 min.
- Discard flow through and place the column back into the collection tube.
- Add 600 µl NT3 buffer and centrifuge at 11.000 g for 1 min.
- Discard flow through and place the column back into the collection tube.
- Centrifuge at 11.000 g for 2 min to remove NT3 buffer. Discard flow through.
- Place the column into a clean 1,5 ml Eppendorf tube.
- Add 30 µl of water or NE buffer and incubate for 1 min to increase the yield of eluted DNA.
- Centrifuge at 11.000 g for 1 min.
Plasmid puriification
This purification is based on the “Zyppy Plasmid Miniprep Kit”
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.
- Initial steps:
- Add 1.5 ml of cell culture in LB medium to 2 ml eppendorf tube.
- Spin at 15.000 g for 2 min.
- Discard supernatant
- Add 1.5 of cell culture (for a total of 3 ml cell culture)
- Spin at 15.000 g for 2 min.
- Remove as much supernatant as possible – pipette carefully. This is (the only) point of no return! To stop, freeze the pellet.
- Add 600 ul of TE-buffer. Ensure that the pellet is completely suspended.
- Add 100 ul 7x lysis buffer. Remember not to process more than 10 minipreps at a time.
- Add 350 ul cold neutralization buffer. Mix gently and thoroughly (= all the way through ≠ violently)!
- Spin at 15.000 g for 5 min.
- 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.
- Spin at 15.000 g for 30 sec.
- Discard flow-through.
- Add 200 ul endo-wash-buffer
- Spin at 15.000 g for 30 sec.
- Add 400 ul zyppy wash buffer
- Spin at 15.000 g for 30 sec.
- 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).
- 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.
- Spin at 15.000 g for 30 sec.
- Check the purification by running a gel (at least until we get experienced with a high success rate).
<Protocol-name-5>
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<Protocol-name-6>
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<Protocol-name-7>
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