Difference between revisions of "Team:UCLA/Notebook/Spider Silk Genetics/2 September 2015"

(Created page with "{{Template:UCLA}} =9/2/2015= ==Sequencing Results== *M1-12(1C3) **1: GOOD **2: Bad, deletion in suffix **3: GOOD *M1-12(T7) **1: incorrect sequence **2: GOOD **3: Bad, no rever...")
 
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**3: Bad, no reverse priming
 
**3: Bad, no reverse priming
 
*M1/2[1:2]-12(1C3)
 
*M1/2[1:2]-12(1C3)
−
**1:
+
**1: GOOD
 +
**2: GOOD
 +
**3: GOOD
 +
*M1/2[1:2]-12(T7)
 +
**1: Bad, no priming
 +
**2: Bad, non-specific
 +
**3: GOOD
 +
 
 +
==Gel Purification for M1-15==
 +
*Used zymo kit.
 +
*Yield 31.64 ng/uL A: 2.47
 +
*Need to do another PCR to get more.
 +
 
 +
==PCR Amplification for M1-15==
 +
*Used 1:1000 dilution of M1-15 from above as template.
 +
**Used ~100 pg of template in reaction.
 +
*Set up 2x 50 uL reactions
 +
{| class="wikitable"
 +
! style="font-weight: bold;" |
 +
! style="font-weight: bold;" | Volume (uL)
 +
|-
 +
| 5x Q5 Buffer
 +
| 10
 +
|-
 +
| 10 mM dNTPs
 +
| 1
 +
|-
 +
| 10 uM For (F-03)
 +
| 2.5
 +
|-
 +
| 10 uM Rev (G-03)
 +
| 2.5
 +
|-
 +
| Template (~30 pg/uL)
 +
| 3j
 +
|-
 +
| Q5 Polymerase
 +
| 0.5
 +
|-
 +
| ddH2O
 +
| 30.5
 +
|-
 +
| style="font-weight: bold;" | Total
 +
| style="font-weight: bold;" | 50
 +
|}
 +
{| class="wikitable"
 +
| 98 C
 +
| 30 sec
 +
|-
 +
| 98 C
 +
| 10 sec
 +
|-
 +
| 66 C
 +
| 20 sec
 +
|-
 +
| 72 C
 +
| 40 sec
 +
|-
 +
| repeat from step 2
 +
| 25x
 +
|-
 +
| 72 C
 +
| 2 min
 +
|-
 +
| 12 C
 +
| hold
 +
|}

Revision as of 21:23, 6 September 2015

iGEM UCLA




9/2/2015

Sequencing Results

  • M1-12(1C3)
    • 1: GOOD
    • 2: Bad, deletion in suffix
    • 3: GOOD
  • M1-12(T7)
    • 1: incorrect sequence
    • 2: GOOD
    • 3: Bad, no reverse priming
  • M1/2[1:2]-12(1C3)
    • 1: GOOD
    • 2: GOOD
    • 3: GOOD
  • M1/2[1:2]-12(T7)
    • 1: Bad, no priming
    • 2: Bad, non-specific
    • 3: GOOD

Gel Purification for M1-15

  • Used zymo kit.
  • Yield 31.64 ng/uL A: 2.47
  • Need to do another PCR to get more.

PCR Amplification for M1-15

  • Used 1:1000 dilution of M1-15 from above as template.
    • Used ~100 pg of template in reaction.
  • Set up 2x 50 uL reactions
Volume (uL)
5x Q5 Buffer 10
10 mM dNTPs 1
10 uM For (F-03) 2.5
10 uM Rev (G-03) 2.5
Template (~30 pg/uL) 3j
Q5 Polymerase 0.5
ddH2O 30.5
Total 50
98 C 30 sec
98 C 10 sec
66 C 20 sec
72 C 40 sec
repeat from step 2 25x
72 C 2 min
12 C hold