User:Derek J. Sanchez/Notebook/TB Sensor - MS applied project/2012/03/08: Difference between revisions

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Find the size of expected part and plasmid back bone and compare to chemical ruler. <br>  
Find the size of expected part and plasmid back bone and compare to chemical ruler. <br>  
<br>  
<br>  
Gel was removed form case and placed on UV viewing stand, and a picture was taken.




==Results (Will be edited with my results by no later than 3/9/2012, current info is property of Dr. Haynes.==


----
'''Minipreps'''<br>
> Check KAH213-218 with E/P<br>
> Check KAH207-209/V0200 with N/S
{| class="wikitable" border="0" cellspacing="3" <!-- Digest check rxn. table -->
|-valign="top"
| <u>Reagent</u> || <u>Volume</u>
| rowspan="7" | <u>Expected:</u><br>V0120 = ~3200<br>1,2. KAH213 = 2834<br>3,4. KAH214 = 1757<br>5,6. KAH215 = 2057<br>7,8. KAH216 = 2590<br>9,10. KAH217 = 1513<br>11,12. KAH218 = 1813<br><br><br>V0200 = 4442<br>13,14. KAH207/V0200 = 1836<br>15,16. KAH208/V0200 = 1884<br>17,18. KAH209/V0200 = 1932
| rowspan="7" | [[Image:KAH_071311_gel1.tif|300px|Miniprep digests]]<br>15 μL/lane; 1% agarose<br><br>[[Image:KAH_071311_gel2.tif|160px|Miniprep digests]]<br>15 μL/lane; 1% agarose
|-
| DNA(plasmid) || 2.0 μL
|-
| 10X buffer || 1.5
|-
| EcoRI || 1.0
|-
| PstI || 1.0
|-
| dH<sub>2</sub>O || 9.5
|-
| &nbsp; || 15 μL --> 37°C/ ~15 min.
|}
----


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Revision as of 13:24, 8 March 2012

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03/08/12

Checking the size of created DNA

The DNA samples prepared yesterday today will be tested for size with electrophoreses.

Restriction Enzyme Digest

Supplies

  • 3μL from each DNA mini prep
  • 1μL Enzym I (EcoR I)
  • 1μL Enzym II (Pst I)
  • 1.5μL 10x Buffer solution (enough for 1x of total volume(15μL))
  • 8.5μL dH2O (enough to get total volume 15μL)
  • One 0.5mL tube per sample


Directions

  1. Create enzyme solution.
    Add chemicals together (except DNA) into master mix
    Add dH2O first
  2. Add 12μL (15μL-3μL of DNA) to labeled 0.5mL tube.
  3. Add 3μL of DNA
  4. Put tubes into hotplate for 10 minutes at 37°C


While this was running an agarose gel was created.
Supplies

Directions

  1. Assemble agarose tray.
    Slide tray into holder ensuring that gasket is in place and flush with bottom.
  2. Insert teeth
    Small teeth, thin up to 15μL thick 20μL
  3. Create agarose solution
    1. Locate agarose flask and fill to 60mL with agarose solution.
    2. Add agarose gelling powder to make 1% solution. (0.1g per 10mL)
    3. Swirl to mix.
    4. Microwave for 40s, and swirl mixture until fully dissolved.
    5. Microwave for 40S.
      Will be hot, may boil over if handled immediately.
    6. Swirl mixture intill no gels/solids are visible.
    7. Add 6μL of Ethidium Bromide (1μL per 10mL).
      Carcinogen avoid skin contact.
  4. Pour solution into tray.
  5. Allow to solidified for 15min.
  6. Carefully remove teeth and tray.
  7. Insert tray into electrophoresis shell
  8. Fill shell to Fill line.
  9. Fill lane one with "chemical ruler"
  10. Add sample to rows and record the position
    Lane 1 = Ruler
    Lane 2 = Sample 1
    Lane 3 = Sample 2
    Lane 4 = Sample 3
  11. Attach lid and probes
  12. Set voltage (110 V)
  13. turn on machine and watch check on gel to ensure it does not flow over.


While the gel is going the expected size can be calculated.
Find the size of expected part and plasmid back bone and compare to chemical ruler.

Gel was removed form case and placed on UV viewing stand, and a picture was taken.


Results (Will be edited with my results by no later than 3/9/2012, current info is property of Dr. Haynes.

Minipreps
> Check KAH213-218 with E/P
> Check KAH207-209/V0200 with N/S

Reagent Volume Expected:
V0120 = ~3200
1,2. KAH213 = 2834
3,4. KAH214 = 1757
5,6. KAH215 = 2057
7,8. KAH216 = 2590
9,10. KAH217 = 1513
11,12. KAH218 = 1813


V0200 = 4442
13,14. KAH207/V0200 = 1836
15,16. KAH208/V0200 = 1884
17,18. KAH209/V0200 = 1932 		Miniprep digests
15 μL/lane; 1% agarose

Miniprep digests
15 μL/lane; 1% agarose
DNA(plasmid) 2.0 μL
10X buffer 1.5
EcoRI 1.0
PstI 1.0
dH2O 9.5
  15 μL --> 37°C/ ~15 min.


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