Team:HokkaidoU Japan/Notebook/August23

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(Difference between revisions)
(→Ligation Negative Control)
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*Used 2 uL of from 100 uL of gel extracted DNA for liagation
*Used 2 uL of from 100 uL of gel extracted DNA for liagation
** Namely added 2 uL of Ligation Solution to 2 uL of DNA
** Namely added 2 uL of Ligation Solution to 2 uL of DNA
-
*Incubated at 16C for 30min
+
*Incubated at 16C for 30 min
===Ligation Negative Control===
===Ligation Negative Control===

Revision as of 07:43, 27 October 2010

Notebook

pSB1C3 Activity Check

EcoR I/Pst I Digestion

Reagent Amount
pSB1C3 50 uL
DW 4 uL
0.1% BSA 7 uL
10x M Buffer 7 uL
EcoR I 1 uL
Pst I 1 uL
Total 70 uL
  • Incubated at 37C for 60 min
  • Added 15 uL of 6x Sample Buffer and electrophoresed on six lanes
  • Extracted from gel

Ligated

  • Used 2 uL of from 100 uL of gel extracted DNA for liagation
    • Namely added 2 uL of Ligation Solution to 2 uL of DNA
  • Incubated at 16C for 30 min

Ligation Negative Control

Used 2 uL of from 100 uL of gel extracted DNA for Transformation

Vector PCR: We meet again!

Electrophoresis to check if PCR was succesful

Amplified pSB1A3, pSB1C3 and pSB1T3 by PCR as listed in the table bellow.

Reagent Amount
Vector 1
DW 33
10x Buffer 5
2 M 4dNTPs 5
25 mM MgSO4 3
Suffix-F 1
Prefix-R 1
KOD 1
Total 50 uL
  • Only deviation from protocol was increase to extention to 120 sec
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