Standard Chlamydomonas reinhardtii PCR_13697068

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title: "TheNessLab : Standard Chlamydomonas reinhardtii PCR"

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2. The Ness Lab Wiki
3. Chlamydomonas information and protocols

TheNessLab : Standard Chlamydomonas reinhardtii PCR

Created by Robert Ness, last modified by Shanza Ayub on Feb 05, 2019

PCR reaction recipe

Ingredient	Concentration	Amount in 20µL PCR (µL)	Final Concentration
ddH2O	-	10.8	-
Buffer	5× 1,2	4.0	1×
MgCl2	25mM	2.0	2.5 mM
dNTP	10mM	1.0	0.5 mM
Primer 1	10µM	0.5	0.25 µM
Primer 2	10µM	0.5	0.25 µM
Taq	5units/µL	0.2	1 unit
DNA4	10ng/µL	1	0.5 ng/µL

1. The concentration of buffers varies by vendor - 5× and 10× are most common. Adjust the volume to ensure 1X final concentration and adjust the ddH₂O to match
2. Some buffers have MgCl₂. This protocol is assuming the  MgCl₂-free buffer, usually labelled -MgCl₂
3. The final concentration of MgCl₂ really alters a PCRs behaviour (more Mg means more permissive binding). In reality it can range from about 1-6mM final concentration. 
4. According to the manual, the lab's current Taq DNA polymerase stock (#EP0401) tolerates 4 pg - 0.4 µg DNA per 20 µL PCR reaction. 

PCR Reaction conditions

Stage	Temp	Time	Cycles
Initial denaturation	95oC	60s	1×
Denaturation	95oC	60s	↑ Repeat 35× ↓
Annealing	55-60oC	30s
Extension	72oC	60s
Final Extentsion	72oC	5m	1×

PCR Protocol: 

*All steps below can be performed on a regular lab bench but you should still wear gloves and use sterile techniques.

1. Prepare a Styrofoam box filled with ice. 
2. Prepare mastermix. The standard x** **uL of DNA sample is 1 uL. Before adding each component of the Mastermix, thaw out the solutions and vortex/flick the tubes briefly. 

   1. For PCR reactions with many DNA sample but only using one pair of primers and ***x ***µL of DNA sample:

      Total volume of Mastermix = 19 uL * *N ,
      *where *N = *1.10 * # of reactions needed (or # of DNA samples). This makes sure you prepare an extra 10% volume of Mastermix to account for pipetting errors. 

      Mastermix= ***N ****  (2.0 µL 10X buffer + 2.0 µL 25mM MgCl₂ + 1.0 µL 10 mM dNTP + 0.5 µL 10 µM forward primer + 0.5 µL 10 µM reverse primer + (12.8-x) uL ddH2O)

   2. For PCR reactions with many DNA sample but  using more than one pair of primers and only x µL of DNA sample:

      Total volume of Mastermix = 18 uL * *N ,
      *where *N = *1.10 * # of reactions needed (or # of DNA samples). This makes sure you prepare an extra 10% volume of Mastermix to account for pipetting errors. 

      Mastermix= ***N ****  (2.0 µL 10X buffer + 2.0 µL 25mM MgCl₂ + 1.0 µL 10 mM dNTP + 0.5 µL 10 µM forward primer + 0.5 µL 10 µM reverse primer + (12.8-x) uL ddH2O)

   3. Good luck with the other scenarios!

3. Load x uL of DNA sample onto the wall of each PCR tube so that the drop of DNA sample is visible. Be careful not to load so many samples at once that the drops of DNA dry up before you start the subsequent steps.
4. If you performed Step 2b, add 0.5 µL 10 µM forward primer and 0.5 µL 10 µM reverse primer to the drop of DNA sample in each PCR tube. 
5. Add*** N** * *0.5 uL Taq polymerase stock to the Mastermix. Freeze the Taq polymerase stock at -20C immediately.
6. Vortex the Mastermix briefly. 
7. Load 20-x uL Mastermix into each PCR tube by pipetting it onto the drop of DNA sample from Step 3. Let the Mastermix roll down to the bottom of the tube with the DNA sample. Whenever you complete a PCR tube or a strip of PCR tubes, place it on ice.
8. Centrifuge the tubes briefly using the mini centrifuge if any drops are stuck on the wall in the PCR tubes. 
9. Place the PCR tubes in the thermocycler using "nesslab"→ "basics" program or other customized programs. 
10. Once the cycle is done, pick up the PCR products and leave the lid open until room temperature to prevent condensation buildup. 

Tips

• If you will be doing a lot of PCR, you should prepare your own stocks of dNTPs. Ideally, the stocks should be several small stocks rather than one large stock.
  **
  Y** uL 10 mM dNTP stock = 0.1 * Y uL dNTP *4 dNTPs + 0.6 * Y uL ddH₂O

• You may end the PCR with a long pause at 4ºC if you are picking up the PCR product within a few hours. Do not leave it at 4ºC past those hours, because condensation will accumulate in the thermocycler and break the machine. It is ok to store PCR product at 10ºC overnight.

Document generated by Confluence on May 22, 2024 11:44

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