User:Matt Hartings/Notebook/Photosynthesis/2012/07/11

(Difference between revisions)

Revision as of 11:28, 11 July 2012

Biomaterials Design Lab

Start cloning sperm whale myoglobin gene into pQE-80 vector

Prep primers
1. Forward Primer (5'-AACAGGATCCGTTCTGTCTGAAGGTGAATGGCAG-3') 0.3mg MW=10546.9g/mol
  1. Add 284uL of autoclaved water to forward primer to make 100uM solution
2. Reverse Primer (5'-AAGCTTACCCTGGTAACCCAGTTCTTTGTA-3') 0.32mg MW=9132g/mol
  1. Add 350uL of autoclaved water to reverse primer to make 100uM solution
Prep PCR tube for reaction
1. 37uL of autoclaved water
2. 10uL of 10X Pfusion HF buffer
3. 1uL of dNTPs
4. 1uL forward primer (meant to use 0.5uL)
5. 1uL reverse primer (meant to use 0.5uL)
6. 1uL of WT swMb DNA
7. 0.5uL of Pfusion HF enzyme
8. 50uL of wax
Place in PCR machine
Run DEL program. (Tamra had edited an older program)

@@ Line 11: / Line 11: @@
 ==Objective==
-Learn how to maintain an OpenWetWare Notebook.
+Start cloning sperm whale myoglobin gene into pQE-80 vector
+==PCR Cloning==
+#Prep primers
+##Forward Primer (5'-AACAGGATCCGTTCTGTCTGAAGGTGAATGGCAG-3') 0.3mg MW=10546.9g/mol
+###Add 284uL of autoclaved water to forward primer to make 100uM solution
+## Reverse Primer (5'-AAGCTTACCCTGGTAACCCAGTTCTTTGTA-3') 0.32mg MW=9132g/mol
+###Add 350uL of autoclaved water to reverse primer to make 100uM solution
+#Prep PCR tube for reaction
+##37uL of autoclaved water
+##10uL of 10X Pfusion HF buffer
+##1uL of dNTPs
+##1uL forward primer (meant to use 0.5uL)
+##1uL reverse primer (meant to use 0.5uL)
+##1uL of WT swMb DNA
+##0.5uL of Pfusion HF enzyme
+##50uL of wax
+#Place in PCR machine
+#Run DEL program. (Tamra had edited an older program)