Team:Stanford/Notebook/Lab Work/Week 8

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== 8/16 Monday ==
== 8/16 Monday ==
===Francisco's Notebook===
===Francisco's Notebook===
-
GFP, RFP - terminator ligations
+
'''Promoter - RSID, sRNA ligations'''
 +
Redesigned oligos (contains entire sequence, has restriction enzyme sticky ends)
-
Promoter - RSID, sRNA ligations
 
-
*Redesigned oligos (contains entire sequence, has restriction enzyme sticky ends)
 
===Karina's Notebook===
===Karina's Notebook===
Need to redesign oligos! It seems that there is a sequence of 16 bp of the "rev-comp scar + scar" that is forming homodimers and selfdimers. 16 bp is significant since our annealing region for the primers is ~20 bp, thus a lot of unwanted byproduct is forming in the PCR stitching reaction. Instead of stitching our parts, we will design our oligos so that they are each the full length of the strand and anneal the two strands together. We will also design our oligos so that they anneal with the XbaI and PstI overhangs, enabling us to skip the restriction digest step.  
Need to redesign oligos! It seems that there is a sequence of 16 bp of the "rev-comp scar + scar" that is forming homodimers and selfdimers. 16 bp is significant since our annealing region for the primers is ~20 bp, thus a lot of unwanted byproduct is forming in the PCR stitching reaction. Instead of stitching our parts, we will design our oligos so that they are each the full length of the strand and anneal the two strands together. We will also design our oligos so that they anneal with the XbaI and PstI overhangs, enabling us to skip the restriction digest step.  
Line 17: Line 16:
== 8/17 Tuesday ==
== 8/17 Tuesday ==
===Francisco's Notebook===
===Francisco's Notebook===
-
GFP, RFP - terminator ligations
+
'''Promoter - RSID, sRNA ligations'''
-
 
+
-
Promoter - RSID, sRNA ligations
+
*Redesigned oligos (contains entire sequence, has restriction enzyme sticky ends)
*Redesigned oligos (contains entire sequence, has restriction enzyme sticky ends)
*Inoculated pBad and pLux; two tubes each, 6 ml culture in each tube
*Inoculated pBad and pLux; two tubes each, 6 ml culture in each tube
-
== 8/18 Wednesday ==
+
===Chris's Notebook===
-
===Karina's Notebook===
+
-
Our order for the redesigned oligos came back because of a series of poly-G runs found in the sRNA 2  Reverse and sRNA 2C Reverse. More than 4 G's in a row forms what is called a "guanine tetracomplex" that is highly unstable knot. The poly-G runs are editable and we'll have to edit our designs and resubmit.
+
-
== 8/19 Thursday ==
+
Sequencing
 +
All came back positive, will inoculate
 +
 
 +
Inoculate
 +
E1, E2, E3, F1, F2, J1x2
 +
 
 +
Miniprep
 +
F2620+B0034, I0500+B0034, MstP, PknH, EmbR
 +
 
 +
Ligation Calculation
 +
Mstp: 1600 bp (320 ng)
 +
PknH: 1300 bp (260)
 +
EmbR: 1200 bp (260)
 +
 
 +
RD of F2620+B0034, I0500+B0034, MstP, PknH, EmbR
 +
 
 +
== 8/18 Wednesday ==
===Francisco's Notebook===
===Francisco's Notebook===
'''Promoter - RSID, sRNA ligations'''
'''Promoter - RSID, sRNA ligations'''
Line 40: Line 51:
*Nanodrop Results for comparison (ng/uL):
*Nanodrop Results for comparison (ng/uL):
**pBad 1: 89.1, pBad 2: 85.3, pLux 1: 32.0, pLux 2: 97.3
**pBad 1: 89.1, pBad 2: 85.3, pLux 1: 32.0, pLux 2: 97.3
-
*Digestion of pLux and pBad at E, S (for 3A assembly)
+
Digestion of pLux and pBad at E, S (for 3A assembly)
*Recipe:
*Recipe:
** Used all of pBad 1 and pLux 2 DNA, added water to top off 34 uL
** Used all of pBad 1 and pLux 2 DNA, added water to top off 34 uL
Line 48: Line 59:
**3.0 uL SpeI (added 3:45pm, heat inactivate 9:50am next day)
**3.0 uL SpeI (added 3:45pm, heat inactivate 9:50am next day)
**3.5 uL EcoRI (added 10:15am next day)
**3.5 uL EcoRI (added 10:15am next day)
 +
===Karina's Notebook===
 +
Our order for the redesigned oligos came back because of a series of poly-G runs found in the sRNA 2  Reverse and sRNA 2C Reverse. More than 4 G's in a row forms what is called a "guanine tetracomplex" that is highly unstable knot. The poly-G runs are editable and we'll have to edit our designs and resubmit.
 +
 +
===Chris's Notebook===
 +
 +
Miniprep
 +
 +
E1, E2, E3, F1, F2, J1x2 (failed bc of columns)
 +
 +
Inoculate
 +
 +
E1, E2, E3, F1, F2, J1x2
 +
 +
Ligation
 +
 +
MstP+B0034, PknH+B0034
 +
 +
Transformed
 +
 +
MstP+B0034, PknH+B0034
 +
 +
Arced, so need to do an ERC
 +
 +
== 8/19 Thursday ==
 +
'''Promoter - RSID, sRNA ligations'''
 +
Diagnostic Gel of pLux and pBad
 +
*promoters cut in many places - had 3 or 4 bands.
 +
 +
===Chris's Notebook===
 +
 +
Miniprep
 +
 +
E1, E2, E3, F1, F2, J1x2 (failed bc of columns)
 +
 +
Inoculate
 +
 +
E1, E2, E3, F1, F2, J1x2
 +
 +
Ligation
 +
 +
MstP+B0034, PknH+B0034
 +
 +
Transformed
 +
 +
MstP+B0034, PknH+B0034
 +
 +
Arced, so need to do an ERC
== 8/20 Friday ==  
== 8/20 Friday ==  
 +
===Francisco's Notebook===
 +
'''Promoter - GFP, RFP - terminator ligations'''
 +
 +
Was going to 3A assemble promoters, reporter and terminator - but there is not RBS (it is coming with the RSID)
 +
Anyways, still
 +
Digested GFP-term 1, RFP-term 1 at X, P (2 hour digestion)
 +
*G1 ~113 ng/uL --> 25 uL
 +
*R1 ~114 ng/uL --> 25 uL
 +
 +
Digested promoters at E, S (2 hour digestion)
 +
*pBad 2 85 ng/uL --> 23 uL (only had this much left)
 +
*pLux 1 32 ng/uL --> 23 uL (only had this much left)
 +
 +
Digested J64100 (ccdb, C resistance) at E, P
 +
*69 ng/uL --> 30 uL
 +
 +
===Chris's Notebook===
 +
 +
Sequencing
 +
All came back positive, will inoculate
 +
 +
Inoculate
 +
E1, E2, E3, F1, F2, J1x2
 +
 +
Miniprep
 +
F2620+B0034, I0500+B0034, MstP, PknH, EmbR
 +
 +
Ligation Calculation
 +
Mstp: 1600 bp (320 ng)
 +
PknH: 1300 bp (260)
 +
EmbR: 1200 bp (260)
 +
 +
RD of F2620+B0034, I0500+B0034, MstP, PknH, EmbR
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Latest revision as of 01:03, 27 October 2010

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Contents

8/16 Monday

Francisco's Notebook

Promoter - RSID, sRNA ligations Redesigned oligos (contains entire sequence, has restriction enzyme sticky ends)

Karina's Notebook

Need to redesign oligos! It seems that there is a sequence of 16 bp of the "rev-comp scar + scar" that is forming homodimers and selfdimers. 16 bp is significant since our annealing region for the primers is ~20 bp, thus a lot of unwanted byproduct is forming in the PCR stitching reaction. Instead of stitching our parts, we will design our oligos so that they are each the full length of the strand and anneal the two strands together. We will also design our oligos so that they anneal with the XbaI and PstI overhangs, enabling us to skip the restriction digest step.

To redesign!

8/17 Tuesday

Francisco's Notebook

Promoter - RSID, sRNA ligations

  • Redesigned oligos (contains entire sequence, has restriction enzyme sticky ends)
  • Inoculated pBad and pLux; two tubes each, 6 ml culture in each tube

Chris's Notebook

Sequencing All came back positive, will inoculate

Inoculate E1, E2, E3, F1, F2, J1x2

Miniprep F2620+B0034, I0500+B0034, MstP, PknH, EmbR

Ligation Calculation Mstp: 1600 bp (320 ng) PknH: 1300 bp (260) EmbR: 1200 bp (260)

RD of F2620+B0034, I0500+B0034, MstP, PknH, EmbR

8/18 Wednesday

Francisco's Notebook

Promoter - RSID, sRNA ligations

Redesigned oligos (contains entire sequence, has restriction enzyme sticky ends)

Miniprepped pBad and pLux; used Zippy kit, eluted in 30 uL Qiagen EB (10mM Tris-Cl)

Diagnostic Gel of pBad and pLux miniprep

  • Gel Estimates (ng/uL):
    • pBad 1: 50, pBad 2: 40, pLux 1: 40, pLux 2: 100
  • Nanodrop Results for comparison (ng/uL):
    • pBad 1: 89.1, pBad 2: 85.3, pLux 1: 32.0, pLux 2: 97.3

Digestion of pLux and pBad at E, S (for 3A assembly)

  • Recipe:
    • Used all of pBad 1 and pLux 2 DNA, added water to top off 34 uL
    • (25 uL pBad 1 + 9 uL water, 26 uL pLux 2 + 8 uL water)
    • 5.0 uL BSA
    • 5.0 uL NEBuffer 4
    • 3.0 uL SpeI (added 3:45pm, heat inactivate 9:50am next day)
    • 3.5 uL EcoRI (added 10:15am next day)

Karina's Notebook

Our order for the redesigned oligos came back because of a series of poly-G runs found in the sRNA 2 Reverse and sRNA 2C Reverse. More than 4 G's in a row forms what is called a "guanine tetracomplex" that is highly unstable knot. The poly-G runs are editable and we'll have to edit our designs and resubmit.

Chris's Notebook

Miniprep

E1, E2, E3, F1, F2, J1x2 (failed bc of columns)

Inoculate

E1, E2, E3, F1, F2, J1x2

Ligation

MstP+B0034, PknH+B0034

Transformed

MstP+B0034, PknH+B0034

Arced, so need to do an ERC

8/19 Thursday

Promoter - RSID, sRNA ligations Diagnostic Gel of pLux and pBad

  • promoters cut in many places - had 3 or 4 bands.

Chris's Notebook

Miniprep

E1, E2, E3, F1, F2, J1x2 (failed bc of columns)

Inoculate

E1, E2, E3, F1, F2, J1x2

Ligation

MstP+B0034, PknH+B0034

Transformed

MstP+B0034, PknH+B0034

Arced, so need to do an ERC

8/20 Friday

Francisco's Notebook

Promoter - GFP, RFP - terminator ligations

Was going to 3A assemble promoters, reporter and terminator - but there is not RBS (it is coming with the RSID) Anyways, still Digested GFP-term 1, RFP-term 1 at X, P (2 hour digestion)

  • G1 ~113 ng/uL --> 25 uL
  • R1 ~114 ng/uL --> 25 uL

Digested promoters at E, S (2 hour digestion)

  • pBad 2 85 ng/uL --> 23 uL (only had this much left)
  • pLux 1 32 ng/uL --> 23 uL (only had this much left)

Digested J64100 (ccdb, C resistance) at E, P

  • 69 ng/uL --> 30 uL

Chris's Notebook

Sequencing All came back positive, will inoculate

Inoculate E1, E2, E3, F1, F2, J1x2

Miniprep F2620+B0034, I0500+B0034, MstP, PknH, EmbR

Ligation Calculation Mstp: 1600 bp (320 ng) PknH: 1300 bp (260) EmbR: 1200 bp (260)

RD of F2620+B0034, I0500+B0034, MstP, PknH, EmbR

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