Team:SCAU-China/Protocol
PCR is used to rapidly amplify certain segments of DNA that are selected via designed primers, with restriction cut sites at the ends of them.
Reagent:
DNA Polymerase (KOD -Plus- Neo)(1.0 U/μl) , 10× PCR Buffer for KOD -Plus- Neo, 25 mM MgSO4, 2 mM dNTPs and Primers.
Step:
1. Preparation of reagent and reaction volume (following volume)
Volume for 25uL reaction
| Purified Template | 0.5uL |
| 10xPCR Buffer | 2.5uL |
| 25mM MgSO4 | 2uL |
| 2mM dNTPs | 2uL |
| Primers | 0.5uL |
| KOD -Plus- Neo | 0.5uL |
| ddH2O | 0.5uL |
Note: 10% DMSO can add to the reaction volume when we want to get a more specific result .
Add the volume to a PCR tube and preset the PCR machine.
2. Sample reaction
We amplify the most of DNA segments by following PCR program:
Predenature : 94℃, 2 min.
Denature : 98℃, 10 sec.
Annealing : (Tm)℃, 30 sec.
Extension : 68℃, 30 sec./ kb
Note:Tm value of primers are higher than 63 ℃ , we use Two Step way :
Predenature : 94℃, 2 min.
Denature : 98℃, 10 sec.
Extension : 68℃, 30 sec./ kb
Note: If the number of copies of the destination fragment is small or expanded, we will try 30-45 cycle.
3. PCR product recovery
We Use the PCR product recovery kit to recycle the DNA segments.
We use the colony-PCR to find out the E.coli carries the correct vector we want.
Reagent:
2xTaq master mixture, Primers
Sept:
1. Preparation of colony-PCR reaction mix volume
We add the reagent totally 360uL (following volume) in a EP tube and divided into 24 tubes.
Volume for 15uL x 24 reaction
| 2x Taq master mixture | 7.5uL x 24 |
| Primers | 0.5uL x 24 |
| ddH2O | 5.5uL x 24 |
2. Pick up a signal colony in the plate with a sterile toothpick and put it in a PCR tube with 15uL reagent. Keep the toothpick in reagent about 3-5 min.
3. Repeat sept2 22 times.
4. For other two tubes, one add 1uL water as the negative control and other one add 1uL DNA template that can amplify the band with a correct size as the active control.
5. Run a PCR and analyze the product, if any sample shows a band with the correct size like the active control, then use this sample for further experiments.
Note:To confirm the colony-PCR result, we will send the sample to sequencing.
School's name:SCAU
Member's name:SCAU
Designed by:SCAU