Minnesota/27 July 2008

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|'''1. Spec the plasmid prep products:''' Place 10 samples into 10 separate plate wells. 11 wells total were used: 1st well was the control so it had 36uL of distilled water and 4uL of elution buffer (since want everything except DNA to be control group), then 2-11 wells had 1-10 plasmid prep products. 2-11 wells had 36uL of distilled water added and 4uL of plasmid prep products. Results:
|'''1. Spec the plasmid prep products:''' Place 10 samples into 10 separate plate wells. 11 wells total were used: 1st well was the control so it had 36uL of distilled water and 4uL of elution buffer (since want everything except DNA to be control group), then 2-11 wells had 1-10 plasmid prep products. 2-11 wells had 36uL of distilled water added and 4uL of plasmid prep products. Results:
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|a. (1) Lac I #1 = [DNA] ng/uL is 45, [DNA] ug/uL is 0.045    HIGH/GOOD
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|'''a.''' (1) Lac I #1 = [DNA] ng/uL is 45, [DNA] ug/uL is 0.045    ''HIGH/GOOD''
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|b. (2) Lac I = [DNA] ng/uL is 35, [DNA] ug/uL is 0.035    HIGH/GOOD
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|'''b.''' (2) Lac I = [DNA] ng/uL is 35, [DNA] ug/uL is 0.035    ''HIGH/GOOD''
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|c. (3) Lac I = [DNA] ng/uL is 35, [DNA] ug/uL is 0.035    HIGH/GOOD
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|'''c.''' (3) Lac I = [DNA] ng/uL is 35, [DNA] ug/uL is 0.035    ''HIGH/GOOD''
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|d. (4) BV:Lac #2 = [DNA] ng/uL is 10, [DNA] ug/uL is 0.001  LOW/POOR
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|'''d.''' (4) BV:Lac #2 = [DNA] ng/uL is 10, [DNA] ug/uL is 0.001  ''LOW/POOR''
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|e. (5) BV:Tet #1 = [DNA] ng/uL is 0, [DNA] ug/uL is 0.00     NONE/BAD
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|'''e.''' (5) BV:Tet #1 = [DNA] ng/uL is 0, [DNA] ug/uL is 0.00     '' NONE/BAD''
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|f. (6) BV:Tet #2 = [DNA] ng/uL is 5, [DNA] ug/uL is 0.005   LOW/POOR
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|'''f.''' (6) BV:Tet #2 = [DNA] ng/uL is 5, [DNA] ug/uL is 0.005   '' LOW/POOR''
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|g. (7) BV:Tet/p22 #1 =
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|'''g.''' (7) BV:Tet/p22 #1 = [DNA] ng/uL is 5, [DNA] ug/uL is 0.005  '' LOW/POOR''
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|'''h.''' (8) BV:Tet/p22 #1 = [DNA] ng/uL is 5, [DNA] ug/uL is 0.005    ''LOW/POOR''
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|'''i.''' (9) BV:Tet/p22 #2 = [DNA] ng/uL is 335, [DNA] ug/uL is 0.335    ''HIGH/GOOD''
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|'''j.''' (10) BV:Tet/p22 #2 = [DNA] ng/uL is 15, [DNA] ug/uL is 0.015    ''LOW/POOR''
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|'''2. Digest Rxn:''' Using #'s: 1, 2, 6, 9, and 10 from above. Also using: p22cII, LAMBDAcI, and RFP from different plasmid prep days. After following table below, allow to incubate for 2 hours. Heat inactivate enzymes @ 65C for 15 minutes in water bath. Follow the table below:
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{|border="1" align="left"
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|-
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!| Parts !! 10x Buffer !! BSA !! DNA !! RE 1 !! RE 2 !! H20
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| BV:Lac I (1) || 5.0uL || 0.5uL || 22.0uL || Spe1, 1.0uL || Pst1, 1.0uL || 20.5uL
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|-
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| BV:Lac I #2 (2) || 5.0uL || 0.5uL || 28.5uL || Spe1, 1.0uL || Pst1, 1.0uL || 14.0uL
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|-
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| BV:Tet  (6) || 5.0uL || 0.5uL || 35.0uL || Spe1, 1.0uL || Pst1, 1.0uL || 7.5uL
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|-
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| BV:Tet/p22 #1 (9) || 5.0uL || 0.5uL || 3.0uL || Spe1, 1.0uL || Pst1, 1.0uL || 39.5uL
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|-
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| BV:Tet/p22 #2 (10) || 5.0uL || 0.5uL || 33.0uL || Spe1, 1.0uL || Pst1, 1.0uL || 9.5uL
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|-
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| p22cII || 5.0uL || 0.5uL || 5.0uL || Xba1, 1.0uL || Pst1, 1.0uL || 37.5uL
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|-
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| LAMBDAcI || 5.0uL || 0.5uL || 7.0uL || Xba1, 1.0uL || Pst1, 1.0uL || 35.5uL
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|-
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| RFP || 5.0uL || 0.5uL || 4.0uL || Xba1, 1.0uL || Pst1, 1.0uL || 38.5uL
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|}
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|'''3. Vector Dephosphorylate:''' Dephosphorylate the base vectors of the digested products. After following the table below, allow to incubate for 30 minutes @37C. Place samples in water bath @ 65C to deactivate the enzyme. Place samples in freezer @ -20C O/N to continue Ligation. Follow the table below:
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{|border="1" align="left"
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|-
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!| Parts !! Antarctic Phosphatase !! Antarctic Phosphatase Buffer
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|-
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| BV:LacI #1 (1) || 1.0uL || 5.0uL
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| BV:LacI #2 (2) || 1.0uL || 5.0uL
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| BV:Tet (6) || 1.0uL || 5.0uL
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| BV:Tet/p22 #1 (9)|| 1.0uL || 5.0uL
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| BV:Tet/p22 #2 (10) || 1.0uL || 5.0uL
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|}

Latest revision as of 20:52, 29 July 2008

Back to Notebook Home
Go to Previous Day (July 26)Go to Next Day (July 28)
1. Spec the plasmid prep products: Place 10 samples into 10 separate plate wells. 11 wells total were used: 1st well was the control so it had 36uL of distilled water and 4uL of elution buffer (since want everything except DNA to be control group), then 2-11 wells had 1-10 plasmid prep products. 2-11 wells had 36uL of distilled water added and 4uL of plasmid prep products. Results:
a. (1) Lac I #1 = [DNA] ng/uL is 45, [DNA] ug/uL is 0.045 HIGH/GOOD
b. (2) Lac I = [DNA] ng/uL is 35, [DNA] ug/uL is 0.035 HIGH/GOOD
c. (3) Lac I = [DNA] ng/uL is 35, [DNA] ug/uL is 0.035 HIGH/GOOD
d. (4) BV:Lac #2 = [DNA] ng/uL is 10, [DNA] ug/uL is 0.001 LOW/POOR
e. (5) BV:Tet #1 = [DNA] ng/uL is 0, [DNA] ug/uL is 0.00 NONE/BAD
f. (6) BV:Tet #2 = [DNA] ng/uL is 5, [DNA] ug/uL is 0.005 LOW/POOR
g. (7) BV:Tet/p22 #1 = [DNA] ng/uL is 5, [DNA] ug/uL is 0.005 LOW/POOR
h. (8) BV:Tet/p22 #1 = [DNA] ng/uL is 5, [DNA] ug/uL is 0.005 LOW/POOR
i. (9) BV:Tet/p22 #2 = [DNA] ng/uL is 335, [DNA] ug/uL is 0.335 HIGH/GOOD
j. (10) BV:Tet/p22 #2 = [DNA] ng/uL is 15, [DNA] ug/uL is 0.015 LOW/POOR
2. Digest Rxn: Using #'s: 1, 2, 6, 9, and 10 from above. Also using: p22cII, LAMBDAcI, and RFP from different plasmid prep days. After following table below, allow to incubate for 2 hours. Heat inactivate enzymes @ 65C for 15 minutes in water bath. Follow the table below:


Parts 10x Buffer BSA DNA RE 1 RE 2 H20
BV:Lac I (1) 5.0uL 0.5uL 22.0uL Spe1, 1.0uL Pst1, 1.0uL 20.5uL
BV:Lac I #2 (2) 5.0uL 0.5uL 28.5uL Spe1, 1.0uL Pst1, 1.0uL 14.0uL
BV:Tet (6) 5.0uL 0.5uL 35.0uL Spe1, 1.0uL Pst1, 1.0uL 7.5uL
BV:Tet/p22 #1 (9) 5.0uL 0.5uL 3.0uL Spe1, 1.0uL Pst1, 1.0uL 39.5uL
BV:Tet/p22 #2 (10) 5.0uL 0.5uL 33.0uL Spe1, 1.0uL Pst1, 1.0uL 9.5uL
p22cII 5.0uL 0.5uL 5.0uL Xba1, 1.0uL Pst1, 1.0uL 37.5uL
LAMBDAcI 5.0uL 0.5uL 7.0uL Xba1, 1.0uL Pst1, 1.0uL 35.5uL
RFP 5.0uL 0.5uL 4.0uL Xba1, 1.0uL Pst1, 1.0uL 38.5uL
3. Vector Dephosphorylate: Dephosphorylate the base vectors of the digested products. After following the table below, allow to incubate for 30 minutes @37C. Place samples in water bath @ 65C to deactivate the enzyme. Place samples in freezer @ -20C O/N to continue Ligation. Follow the table below:


Parts Antarctic Phosphatase Antarctic Phosphatase Buffer
BV:LacI #1 (1) 1.0uL 5.0uL
BV:LacI #2 (2) 1.0uL 5.0uL
BV:Tet (6) 1.0uL 5.0uL
BV:Tet/p22 #1 (9) 1.0uL 5.0uL
BV:Tet/p22 #2 (10) 1.0uL 5.0uL
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