Difference between revisions of "Team:SJTU-BioX-Shanghai/Protocol"
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</tr> | </tr> | ||
</tbody></table> | </tbody></table> | ||
| + | ==PCR== | ||
| + | ===Primer Design=== | ||
| + | ====Several principles should be followed in primer designing==== | ||
| + | <html> | ||
| + | <p> | ||
| + | |||
| + | 1. The length of primers should be limited between 20~60bp. For overlapping PCR, the overlapping domain should contains at least 15bp.</br> | ||
| + | 2. GC% should be at around 40%~60%.</br> | ||
| + | 3. Before synthesis, blast the primer using software like Vector NTI or other methods to check the specificity.</br> | ||
| + | The primers are synthesized by GeneWise. After the arriving of the primers, perform a quick spin and then dissolve the DNA powder using ddwater in the given volume.</br> | ||
| + | PCR Volume:</br> | ||
| + | If the PCR product will be used for testing, we suggest the volume should be under 20 μl. If the PCR product will be used for any downstream operation (eg. Purification, gel extraction, restriction enzyme digestion), higher volume such as 50μl should be used. And for every sample, there should be at least two parallels. So, mix the total mixture in a 1.5ml centrifuge tube beforehand and then separate the desired volume mixture into each PCR tube is recommended.</br> | ||
| + | PCR Reaction Mixture:</br> | ||
| + | We used 2×PrimeSTAR Mix from TAKARA and 2×Taq Master Mix from LifeFeng for PCR reaction, which has already contained buffer, magnesium, dNTP and polymerase. The amount of primer should be at around 10μM and the total amount of template should be under 200ng as protocol required (less template is needed if use genomic DNA or plasmid DNA as template, see specifically in the protocols of the products). Additionally, the bacterial liquid template must not be too much or else bacteria may cause a jam when performing gel analysis. And pay attention to the overlapping PCR: the amount of the two templates should be equal and the primer is added later (see specifically at the example “Overlapping PCR”).</br> | ||
| + | PCR Conditions:</br> | ||
| + | Denaturing conditions-94℃～98℃ for 5~15 seconds. For bacterial liquid PCR, longer denaturing time (10~15minutes) is required for completely cell splitting.</br> | ||
| + | Annealing temperature-the annealing temperature is usually 3~5℃ lower than the melting temperature of primers. If the melting temperatures of the two primers differ greatly (for example, >5℃), try gradient PCR to find out the appropriate annealing temperature.</br> | ||
| + | Here are examples of the three kinds of PCR that we used in the experiments:</br> | ||
| + | General PCR (50μl volume): | ||
| + | |||
| + | <p> | ||
</html> | </html> | ||
{{ SJTU-BioX-Shanghai/Footer }} | {{ SJTU-BioX-Shanghai/Footer }} | ||
{{ SJTU-BioX-Shanghai/Style }} | {{ SJTU-BioX-Shanghai/Style }} | ||
Revision as of 00:48, 11 September 2015
Contents
Algae transformation
BG11 medium
The content of mother solution
|
Mother liquors |
substance |
content(g/L) |
For 100mL （g） |
|
1 |
K2HPO4·3H2O |
40 |
4 |
|
2 |
MgSO4·7H2O |
75 |
7.5 |
|
3 |
Ferric ammonium citrate |
6 |
0.6 |
|
Citrate acid |
6 |
0.6 |
|
|
4 |
CaCl2·2H2O |
36 |
3.6 |
|
5 |
EDTA-Na2 |
1 |
0.1 |
|
6 |
H3BO3 |
2.86 |
0.286 |
|
MnCl2·4H2O |
1.81 |
0.181 |
|
|
ZnSO4·7H2O |
0.222 |
0.0222 |
|
|
NaMoO4·5H2O |
0.39 |
0.039 |
|
|
CuSO4·5H2O |
0.079 |
0.0079 |
|
|
Co（NO3）2·6H2O |
0.04 |
0.004 |
Liquid medium (for 1L)
1ml of each mother liquors, besides, add
|
NaNO3 |
1.5g |
|
Na2CO3 |
0.02g |
Adjust pH to 7.5-7.55, 121℃ autoclaved for 20mins.
Solid medium (for 1L)
1ml of each mother liquors, besides, add
|
NaNO3 |
1.5g |
|
Na2CO3 |
0.02g |
|
Na2S2O3 |
3g |
|
TES |
2.22 |
|
Agar |
1.5% Which means 15g |
Adjust pH to 7.5-7.55,121℃ autoclaved for 20mins. Prepare solid plate before use. Be sure the antibiotic is active and in appropriate concentration.
Transformation of hunger
|
Time |
Steps |
|
Day 1 at noon |
Set the culture，the initial OD730 is 0.1 |
|
Day 2 in the evening |
After 1.5 days，the OD730 would approach to about 0.5（even though 0.3-0.7 is ok）. |
|
Centrifuge at RT*3000g*10min，remove the supernatant，add 1ml liquid medium，measure the od730 by nanodrop, then calculate the volume of cyanobacteria to adjust the ultimate od730 to 2.5. |
|
|
Calculate the volume of DNA（5-10ug，usually take 8ug） |
|
|
Mix the DNA, cyanobacteria and liquid medium in a 15 ml centrifuge tube to make the total volume is 500ul and the od730 is about 2.5, set a negative control (without DNA). |
|
|
Overnight and then shake slightly by hand for about 4 times.（every 2-3 hours） |
|
|
Day 3 at afternoon |
Take 200ul culture, spread it on solid plate with suitable antibiotic, put the plate into illumination incubator at 30℃. |
Then wait for several days (depends on the gene)
If the solid plate with DNA have colony while the negative plate is clean, then we may conclude the natural transformation is successful, but we need three experiments for further determination.
|
1. |
Pick up the colony，spread it in a solid plate (with antibiotic) in parallel, put it in illumination incubator to store the colony. (Not all the colony can grow because this can work as segregation.) |
|||||||||||||||||||||||||
|
2 |
Pick up the colony |
Set a 500ul liquid culture, then add it into an 100ml flask for further segregation. |
||||||||||||||||||||||||
|
Spread it on solid plate in parallel to store the cyanobacteria. |
||||||||||||||||||||||||||
|
Set a 50ul liquid culture to do colony PCR |
||||||||||||||||||||||||||
|
|
Colony PCR：put the 50ul liquid culture into liquid N2, then into 80℃ water, repeat these steps for another 2 times, take 0.5ul to do colony PCR. PCR system：
PCR program：
Run gel to determination. |
|||||||||||||||||||||||||
Transformation of electricity
|
Day 1 at noon |
Set the culture. |
|
Prepare the electric switch and electric tubes, wash the tubes with ddH2O for several times, then wash with 75% ethanol, put them in a beaker, covering with 75% ethanol. |
|
|
Day 3 in the evening or at the afternoon |
Take out the electric tubes from ethanol, put them into 60℃ oven for at least 2 hours to dry the tube, sterilize them by UV before electrotransformation. |
|
Centrifuge at 8000rpm*5min (the OD730 is about 0.4), dilute the cell with sterilized ddH2O (40,30,30,20,20ml separately) wash it for 5 times. (suggest 7 times at first time) |
|
|
Dilute the cyanobacteria with 200ul sterilized ddH2O, mix it with DNA (at least 500ng) to make the total volume to 50ul，add them into electric tube, tunk for several times so that the liquid can reach to the bottom. |
|
|
Put the electric tube in electric switch correctly, choose Ec1 program, press “pulse”, wait for a moment, check the voltage and time. (The best condition is 12kV & 5ms, but we take 18kV & 4-6ms as well.) Add 1ml liquid BG11 culture immediately, mix and add back to 50ml BG11 culture, put it in shaking illumination incubator at 30℃*130rpm for 1day. |
|
|
Day 4 in the evening |
Collect the cyanobacteria at 8000rpm*5min, dilute the cell with 400ul liquid culture, mixed with 5ml soft agar BG11 medium, spread on solid medium (antibiotic added), put the plate into illumination incubator at 30 ℃. |
Wait for several days (depends on your gene), then test your colony just like natural transformation.
Prove transformation success
Three experiments to prove the success of transformation
|
1. |
Pick up the colony，spread it in a solid plate (with antibiotic) in parallel, put it in illumination incubator to store the colony. (Not all the colony can grow because this can work as segregation.) |
|||||||||||||||||||||||||
|
2 |
Pick up the colony |
Set a 500ul liquid culture, then add it into an 100ml flask for further segregation (at least four generations). |
||||||||||||||||||||||||
|
Spread it on solid plate in parallel to store the cyanobacteria. |
||||||||||||||||||||||||||
|
Set a 50ul liquid culture to do colony PCR |
||||||||||||||||||||||||||
|
|
Colony PCR：put the 50ul liquid culture into liquid N2, then into 80℃ water, repeat these steps for another 2 times, take 0.5ul to do colony PCR. PCR system：
PCR program：
Run gel to determination. |
|||||||||||||||||||||||||
1. The length of primers should be limited between 20~60bp. For overlapping PCR, the overlapping domain should contains at least 15bp. 2. GC% should be at around 40%~60%. 3. Before synthesis, blast the primer using software like Vector NTI or other methods to check the specificity. The primers are synthesized by GeneWise. After the arriving of the primers, perform a quick spin and then dissolve the DNA powder using ddwater in the given volume. PCR Volume: If the PCR product will be used for testing, we suggest the volume should be under 20 μl. If the PCR product will be used for any downstream operation (eg. Purification, gel extraction, restriction enzyme digestion), higher volume such as 50μl should be used. And for every sample, there should be at least two parallels. So, mix the total mixture in a 1.5ml centrifuge tube beforehand and then separate the desired volume mixture into each PCR tube is recommended. PCR Reaction Mixture: We used 2×PrimeSTAR Mix from TAKARA and 2×Taq Master Mix from LifeFeng for PCR reaction, which has already contained buffer, magnesium, dNTP and polymerase. The amount of primer should be at around 10μM and the total amount of template should be under 200ng as protocol required (less template is needed if use genomic DNA or plasmid DNA as template, see specifically in the protocols of the products). Additionally, the bacterial liquid template must not be too much or else bacteria may cause a jam when performing gel analysis. And pay attention to the overlapping PCR: the amount of the two templates should be equal and the primer is added later (see specifically at the example “Overlapping PCR”). PCR Conditions: Denaturing conditions-94℃～98℃ for 5~15 seconds. For bacterial liquid PCR, longer denaturing time (10~15minutes) is required for completely cell splitting. Annealing temperature-the annealing temperature is usually 3~5℃ lower than the melting temperature of primers. If the melting temperatures of the two primers differ greatly (for example, >5℃), try gradient PCR to find out the appropriate annealing temperature. Here are examples of the three kinds of PCR that we used in the experiments: General PCR (50μl volume):
