Difference between revisions of "Team:UCLA/Notebook/Honeybee Silk/30 May 2015"

(Created page with "='''Gel Visualization of 5/26 PCR'''= The 25uL PCR products from 5/26 were run on a 1% agarose gel with 5uL 6X loading dye against a 1kB ladder. (Need to add gel photo) ===Re...")
 
(→‎Gel Visualization of 5/26 PCR)
 
(3 intermediate revisions by one other user not shown)
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<a href="https://2015.igem.org/Team:UCLA/Notebook/Spider_Silk_Genetics"><li>Spider Silk Genetics</li></a>
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<a href="https://2015.igem.org/Team:UCLA/Notebook/Honeybee_Silk"><li>Honeybee Silk</li></a> 
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='''Gel Visualization of 5/26 PCR'''=
 
='''Gel Visualization of 5/26 PCR'''=
  
−
The 25uL PCR products from 5/26 were run on a 1% agarose gel with 5uL 6X loading dye against a 1kB ladder.
+
The PCR products from 5/26 were run on a 1% agarose gel against a 1kB ladder. I used 5 uL of each sample and added 1uL of loading dye.
 +
 
 +
[[File:2015-05-26 gel.jpg|none|thumb|500px|Gel Image]]
  
−
(Need to add gel photo)
 
  
  
Line 9: Line 298:
  
 
There were bands present for the correct base pair length (~1000bp). The sample run at 67C for the annealing step produced the cleanest, brightest band.
 
There were bands present for the correct base pair length (~1000bp). The sample run at 67C for the annealing step produced the cleanest, brightest band.
−
 
  
 
='''PCR of Honeybee G-block (Optimal)'''=
 
='''PCR of Honeybee G-block (Optimal)'''=
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! Initial Denaturation
 
! Initial Denaturation
 
| 98C
 
| 98C
−
| 3 min
+
| 30s
 
|-
 
|-
 
! Cycles (x25)
 
! Cycles (x25)
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| Hold
 
| Hold
 
|}
 
|}
 +
 +
='''Purification of PCR product'''=
 +
 +
I purified my PCR product using Qiagen MinElute columns, following their protocol.
 +
 +
OD: 349.18ng/uL

Latest revision as of 22:32, 16 July 2015

Gel Visualization of 5/26 PCR

The PCR products from 5/26 were run on a 1% agarose gel against a 1kB ladder. I used 5 uL of each sample and added 1uL of loading dye.

Gel Image


Results:

There were bands present for the correct base pair length (~1000bp). The sample run at 67C for the annealing step produced the cleanest, brightest band.

PCR of Honeybee G-block (Optimal)

From the gel visualization, 67C annealing temperature produced the best results. I am running a 50uL PCR reaction now under the optimal conditions.

Component Volume (out of 50uL)
5X Q5 Reaction Buffer 10uL
10mM dNTPS 1uL
10mM primer 10 2.5uL
10mM primer 11 2.5uL
Template (Honeybee G-block) 1uL
Q5 High Fidelity DNA Polymerase 0.5uL
Nuclease Free Water 32.5uL

Program

Step Temperature Time
Initial Denaturation 98C 30s
Cycles (x25) 98C 10s
Annealing 67C 20s
Extension 72C 30s
Final Extension 72C 2min
Hold 12C Hold

Purification of PCR product

I purified my PCR product using Qiagen MinElute columns, following their protocol.

OD: 349.18ng/uL