Minnesota/10 July 2008

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Revision as of 16:07, 11 July 2008

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1. Dephosphorylation: Continued with double digestion base vector product from 07-09-2008. Used Antartic phosphoylase to dephosphorylate the base vector. Want to dephosphorylate the base vector to prevent it from closing - want it to stay a linear digested product. After dephosphorylation, the base vector was then incubated @ 37C for 30 minutes. Sample was then placed in a heat bath @ 65C for 15 minutes to inactivate enzyme (phosphotase).

Gene	10x Buffer	DNA	Phosphotase
Base Vector	5.6uL	50.0uL	1.0uL

NOTE: Total volume in dephosphorylation = 56.6uL

2. Run RXN model on Calhoun (super computer).

3. Dilute base vector culture, allow to grow, then miniprep, then streak plates and make base vector glycerol stocks.

4. Autoclave dishes

5. Ligation: Follow table below. When ligation is complete, incubate products @16C for 1 hour. Heat inactivate DNA ligase (enzyme) @ 65C for 15 minutes.

Reagent	Buffer	H20	Insert DNA	LAMBDAcI DNA	Dephosphor. Base V.	T4 DNA Ligase
pro-LAMBDAcI-B.V.	4.0uL	---	promoter 1.0uL	6.0uL	10.0uL	1.0uL
LAMBDAcI-term-B.V.	4.0uL	---	terminator 1.0uL	6.0uL	10.0uL	1.0uL
Control	4.0uL	7.0uL	---	---	10.0uL	1.0uL

6. Ligation product Transformations: Transform the ligation products to TOP10 E. Coli cells. Allow growth for 2 hours. Following growth, 200 uL of all four ligation reactions and a control were plated on Kanamycin LB and were incubated at 37 degrees overnight.

7. Ordered Primers: Ordered primers for possible use in real-time PCR.

Primer	Sequence
F Mcherry for modification	gaattcgcggccgcttctagatggtgagcaagggcgagg
R Mcherry for modification	TATAAACGCAGAAAGGCCCACCC
R LAMcI for realtime PCR	GGTTGTGCTTACCCATCTCTCC
R p22mnt for realtime PCR	ACTCGCTCTGCTCATCGGCG
R p22cII for realtime PCR	CAATCTACAGTGGTGTCGTGCC
R GFP for realtime PCR	GAATGTTTCCATCTTCTTTAAAATC
R mCherry for realtime PCR	GTGATGAACTTCGAGGACGGCG

@@ Line 32: / Line 32: @@
 {|border="1" align="left"
 |-
-!| Reagent !!Volume in uL
+!| Reagent !! Buffer !! H20 !! Insert DNA !! LAMBDAcI DNA !! Dephosphor. Base V. !! T4 DNA Ligase
 |-
-|10x Buffer ||2.0uL
+|pro-LAMBDAcI-B.V. ||4.0uL || ---||promoter 1.0uL ||6.0uL ||10.0uL ||1.0uL
 |-
-|H20 ||20.0uL
+|LAMBDAcI-term-B.V. ||4.0uL || ---||terminator 1.0uL ||6.0uL ||10.0uL ||1.0uL
 |-
-|Vector ||
+|Control ||4.0uL || 7.0uL || ---|| ---|| 10.0uL ||1.0uL
-|-
-|DNA ||
-|-
-|T4 DNA Ligase || 1.0uL
 |-
 |}