28017399
Created by Safa Ahmad, last modified on Sep 22, 2018
Title (Broad-Range (B) and *C.reinhardtii-*specific (R)) refers to primers used to attempt to identify Chlamy using DNA extracts from algal cultures derived from soil samples.
25 uL PCR Recipe (modified from NEB basic protocol here)
| Reagent | Volume Added/ uL | Final Concentration |
|---|---|---|
| 10x NH4 2+ OR 10x KCl Buffer | 2.5 | 1x |
| 25 mM Mg2+ |
2.5 if NH4 used 1.5 if KCl used |
2.5 mM 1.5 mM |
| Taq Polymerase | 0.125 | 0.625 units/25 uL |
| 10 mM dNTPs | 0.5 | 200 uM |
| 10 uM Reverse Primer | 0.5 | 0.2 uM |
| 10 uM Forward Primer | 0.5 | 0.2 uM |
| 20-25 ng/uL DNA extract | 1 | 1ish ng/uL |
| 5 M Betaine (if required) | 5 | 1 M |
| MilliQ Water | 12.375 |
|
Cycle Conditions
| Temp/ C | Time | Changed Ramp Rate from default (if applicable) - oC /s |
|---|---|---|
| 95 | 3 min |
|
| Cycle Step 1: 95 | 1 min | 1 |
| Cycle Step 2: Annealing Temp (5 C below Melting) | 30 s | 1 |
| Cycle Step 3: 72 | 1 min | 1 |
| 72 | 5 min |
|
Notes on Primers 1R-10R and 1B-9B
| Primer Pairs | Note |
|---|---|
| 2B, 5B, 5R | Have not been tested |
| 7R, 8R | Explicitly worked with KCl buffer. perhaps in need of further optimization if to be used with NH42+ |
| 2R, 4R, 3B, 8B | Pairs selected and used for final stage of detection |
|
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|
Copy of
PCR (with Broad-Range and Rein-Specific Chlamy
Primers).xlsx
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PCR
(with Broad-Range and Rein-Specific Chlamy
Primers).xlsx
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PCR
(with Broad-Range and Rein-Specific Chlamy
Primers).xlsx
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PCR
(with Broad-Range and Rein-Specific Chlamy
Primers).xlsx
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How I
found Broad-Range Primers.docx
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PCR
protocol (with Broad-Range and Rein-Specific Chlamy
Primers).xlsx
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Primer
List (June '18) (1).xlsx
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Primer
Testing.xlsx
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Safa_ROPNessLab2018_Pres.pdf
(application/pdf)
Safa_ROPReport_NessLab2018 (2).pdf
(application/pdf)
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