"
Team:AUC Turkey/Procedures
From 2012hs.igem.org
Selcuk.igem (Talk | contribs) |
Selcuk.igem (Talk | contribs) |
||
| Line 4: | Line 4: | ||
Take the average of the values measured by a spectrophotometer to dna. | Take the average of the values measured by a spectrophotometer to dna. | ||
| + | |||
Divide 500 by DNA average. | Divide 500 by DNA average. | ||
| + | |||
Put 5ul Ne Buffer | Put 5ul Ne Buffer | ||
| - | Put 0.5ul BSA Buffer | + | |
| + | Put 0.5ul BSA Buffer | ||
| + | |||
Add 1 ul of the enzymas with barrier tips | Add 1 ul of the enzymas with barrier tips | ||
| + | |||
İf you cut with EcoR1 and SpI, it will be up stream. | İf you cut with EcoR1 and SpI, it will be up stream. | ||
| + | |||
İf you cut with Xbal and Pst1, it will be down stream. | İf you cut with Xbal and Pst1, it will be down stream. | ||
| + | |||
(Attention ! enzymes shouldn’t heat) | (Attention ! enzymes shouldn’t heat) | ||
| + | |||
Subtract the amount of DNA from 42.5 ul. This result will be the amount of NFW used. | Subtract the amount of DNA from 42.5 ul. This result will be the amount of NFW used. | ||
| + | |||
Add the NFW with barrier tips and do one pippetting while taking the NFW. | Add the NFW with barrier tips and do one pippetting while taking the NFW. | ||
| + | |||
Then the DNA is put into the PCR and is left there for 30 minutes. | Then the DNA is put into the PCR and is left there for 30 minutes. | ||
| + | |||
== Ligation protocol == | == Ligation protocol == | ||
| Line 19: | Line 30: | ||
2ul up stream is put into a eppendorf. | 2ul up stream is put into a eppendorf. | ||
| + | |||
2ul down stream is also added. | 2ul down stream is also added. | ||
| + | |||
2ul plasmid is mixed in as well. | 2ul plasmid is mixed in as well. | ||
| + | |||
2ul Taq Buffer is inserted to the mixture. | 2ul Taq Buffer is inserted to the mixture. | ||
| + | |||
1ul T4 DNA ligase is then added with barrier tips. | 1ul T4 DNA ligase is then added with barrier tips. | ||
| + | |||
11ul NFW is added with barrier tips and should be pipetted once. | 11ul NFW is added with barrier tips and should be pipetted once. | ||
| + | |||
Then the DNA is put into the PCR and is left there for 30 minutes. | Then the DNA is put into the PCR and is left there for 30 minutes. | ||
| - | Gel Preparation | + | |
| + | |||
| + | == Gel Preparation == | ||
| + | |||
| + | |||
Mix 100ml TAE and 0,8 gram agarose in a glass beaker. | Mix 100ml TAE and 0,8 gram agarose in a glass beaker. | ||
| + | |||
The mixture is then heated in a microwave for 3 minutes. | The mixture is then heated in a microwave for 3 minutes. | ||
| + | |||
Afterwards, 3,6 ul EtBr is added and the beaker is mixed on a magnetic mixer. | Afterwards, 3,6 ul EtBr is added and the beaker is mixed on a magnetic mixer. | ||
| + | |||
Mold them and wait for 20 minutes fort he gel to harden. | Mold them and wait for 20 minutes fort he gel to harden. | ||
| Line 35: | Line 59: | ||
Put 3 ul coloring agent on a strand of parafilm. | Put 3 ul coloring agent on a strand of parafilm. | ||
| + | |||
Take 7 ul plasmid and do pipeting with the colouring agent. | Take 7 ul plasmid and do pipeting with the colouring agent. | ||
| + | |||
Switch the pipette to 10 ul and take the colored plasmid. | Switch the pipette to 10 ul and take the colored plasmid. | ||
| + | |||
Place the plasmid into one of the holes of our gel. | Place the plasmid into one of the holes of our gel. | ||
| + | |||
Give electricity to the anode and cathode in required amounts. | Give electricity to the anode and cathode in required amounts. | ||
| + | |||
Wait according to the tank and the amount of electricity. | Wait according to the tank and the amount of electricity. | ||
| + | |||
Use the camera to get the results. | Use the camera to get the results. | ||
<forum_subtle/> | <forum_subtle/> | ||
Revision as of 17:28, 16 June 2012
| Sponsors | ||
|---|---|---|
|
|
|
Digestion protocol
Take the average of the values measured by a spectrophotometer to dna.
Divide 500 by DNA average.
Put 5ul Ne Buffer
Put 0.5ul BSA Buffer
Add 1 ul of the enzymas with barrier tips
İf you cut with EcoR1 and SpI, it will be up stream.
İf you cut with Xbal and Pst1, it will be down stream.
(Attention ! enzymes shouldn’t heat)
Subtract the amount of DNA from 42.5 ul. This result will be the amount of NFW used.
Add the NFW with barrier tips and do one pippetting while taking the NFW.
Then the DNA is put into the PCR and is left there for 30 minutes.
Ligation protocol
2ul up stream is put into a eppendorf.
2ul down stream is also added.
2ul plasmid is mixed in as well.
2ul Taq Buffer is inserted to the mixture.
1ul T4 DNA ligase is then added with barrier tips.
11ul NFW is added with barrier tips and should be pipetted once.
Then the DNA is put into the PCR and is left there for 30 minutes.
Gel Preparation
Mix 100ml TAE and 0,8 gram agarose in a glass beaker.
The mixture is then heated in a microwave for 3 minutes.
Afterwards, 3,6 ul EtBr is added and the beaker is mixed on a magnetic mixer.
Mold them and wait for 20 minutes fort he gel to harden.
ELECTROPHORESIS PROTOCOL
Put 3 ul coloring agent on a strand of parafilm.
Take 7 ul plasmid and do pipeting with the colouring agent.
Switch the pipette to 10 ul and take the colored plasmid.
Place the plasmid into one of the holes of our gel.
Give electricity to the anode and cathode in required amounts.
Wait according to the tank and the amount of electricity.
Use the camera to get the results.
<forum_subtle/>
