Haynes Lab:Notebook/Engineering PC-TFs/2012/12/07: Difference between revisions

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==Summary==
==Summary==
*  
* Performed another PCR run because lid was not closed entirely yesterday. Next time in lab(Monday), confirmation gel will be run to show that PCR rxns worked.




*Assemblies
1. hPCD - Pflex - BL01<br>
2. hPCD - BL01
{|border="1" cellpadding="5" cellspacing="0"
|-
! scope="col" style="background:#efefef;" | #
! scope="col" style="background:#efefef;" | Template
! scope="col" style="background:#efefef;" | Primers
|-
|1
|hPCD
|9, 2
|-
|2
|Plflex
|3, 4
|-
|3
|BL01
|5,10
|-
|4
|hPCD
|9, 7
|-
|5
|BL01
|8,10
|-
|}
{|border="1" cellpadding="5" cellspacing="0"
|-
! scope="col" style="background:#efefef;" |
! scope="col" style="background:#efefef;" | μL
|-
|Template
|.5 μL
|-
|Primer 1 (10 μm)
|1.0 μL
|-
|Primer 2 (10 μm)
|1.0 μL
|-
|2*GoTaq
|12. 5
|-
|DH2O
|10.0 μL
|-
|}
PCR was set with preset settings:
[[Image:pcrsett.jpg|300px]]
----
*Ran Zymoclean DNA recovery kit.
*Add 3 volumes of ADB Buffer to each volume of gel. (Each gel slice is assumed to be 200 mg)
*Therefore, add 600 μL of ADB Buffer.
*Incubate at 55°C for 10 minutes.
*Load melted agarose solution into Spin Column in a collection tube.
*Centrifuge at max speed for 30 seconds.
*Discard flow-through.
*Add 200 μL of DNA wash buffer to the spin columns and centrifuge for 30 seconds.
*Place spin column in a 1.5 mL tube. Add 10μL of DNA Elution Buffer to elute the DNA.
*Place tube into freezer box.
''There was not much DNA that came from the purification kit. A reason for this is because the bands were smaller than usual, according to Vi. Vi is running another gel digest to cut the backbone from the PSB1A3 vector. Rene said she was also running into problems with minipreping the vectors and cutting it. A follow-up with Dr. Haynes will confirm next step and possible options.
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__NOTOC__
__NOTOC__

Revision as of 16:04, 7 December 2012

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Summary

  • Performed another PCR run because lid was not closed entirely yesterday. Next time in lab(Monday), confirmation gel will be run to show that PCR rxns worked.


  • Assemblies

1. hPCD - Pflex - BL01
2. hPCD - BL01

# Template Primers
1 hPCD 9, 2
2 Plflex 3, 4
3 BL01 5,10
4 hPCD 9, 7
5 BL01 8,10
μL
Template .5 μL
Primer 1 (10 μm) 1.0 μL
Primer 2 (10 μm) 1.0 μL
2*GoTaq 12. 5
DH2O 10.0 μL

PCR was set with preset settings:

  • Ran Zymoclean DNA recovery kit.
  • Add 3 volumes of ADB Buffer to each volume of gel. (Each gel slice is assumed to be 200 mg)
  • Therefore, add 600 μL of ADB Buffer.
  • Incubate at 55°C for 10 minutes.
  • Load melted agarose solution into Spin Column in a collection tube.
  • Centrifuge at max speed for 30 seconds.
  • Discard flow-through.
  • Add 200 μL of DNA wash buffer to the spin columns and centrifuge for 30 seconds.
  • Place spin column in a 1.5 mL tube. Add 10μL of DNA Elution Buffer to elute the DNA.
  • Place tube into freezer box.

There was not much DNA that came from the purification kit. A reason for this is because the bands were smaller than usual, according to Vi. Vi is running another gel digest to cut the backbone from the PSB1A3 vector. Rene said she was also running into problems with minipreping the vectors and cutting it. A follow-up with Dr. Haynes will confirm next step and possible options.


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