Difference between revisions of "Team:UCLA/Notebook/Spider Silk Genetics/30 August 2015"

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=8/27/2015=
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=8/30/2015=
  
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==Sequencing Results==
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==Gel Purification==
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*M1-12(1C3)
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*Used zymo kit to purify gels of M1-12 and M1/2[1:2]-12, elution in 12 uL
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**1: wrong sequence entirely
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*M1-12: 42.79 ng/uL
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**2: wrong sequence entirely
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*M1/2[1:2]-12: 57.8 ng/uL
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**3: wrong sequence entirely
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**Interestingly, all three sequences align to each other.
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==Glycerol Stock Reconstitution==
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*M1/2[1:1]-12(1C3)
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*Plated M1/2[1:1]-12(T7), M1/2[2:1]-12(T7) and M1-SeqAB2 on LB + chlor plates.
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**1: Bad, single base pair mutation at ~550
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**2: GOOD
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==RE digestion for M1-12, M1/2[1:2]-12==
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**3: GOOD
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*Digested M1-12, M1/2[1:2]-12 each with E,P and X,P.
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*M1/2[1:1]-12(T7)
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*Digest 5 uL of the samples purified today in a 50 uL reaction with 1 uL each of the respective enzymes.
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**1: Bad, deletion at ~80
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*Digest 37 C for 1.5 hrs, heat at 65 C for 20 min.
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**2: GOOD
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*Digests were purified using Zymo clean and concentrator.
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**3: possibly correct, but not trustworthy due to short read.
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*M1/2[2:1]-12(1C3)
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==Ligation==
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**1: Bad, single bp mutation near ~660
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*For a 3:1 ligation to 50 ng of vector, 96 ng of 1324 bp insert is required
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**2: Bad, single bp mutation at 962
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*For a 5:1 ligation to 50 ng of vector, 160 ng of 1324 bp insert is required.
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**3: GOOD
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*Ligated E,P digested products into pSB1C3.
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*M1/2[2:1]-12(T7)
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*Ligated X,P digested products into BBaK_525998.
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**1: bad, non-silent single bp mutation at 1004
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*For M1-12 ligations, used 10 uL of digested product to approximate a 3:1 insert:vector ratio due to insufficient amounts.
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**2: GOOD? silent single bp mutation at 702
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*For M1/2[1:2]-12 ligations, used enough product to ligate at a 5:1 insert:vector ratio.
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**3: GOOD? silent single bp mutation at 702
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*Ligate at 25 C for 1 hr, heat kill at 65 C for 20 min.
 +
 
 +
==Transformation==
 +
*Transform M1-12(1C3) and M1/2[1:2]-12(1C3) into DH%(alpha) electrocompetent cells.
 +
**Ligation products were dialyzed against ultra-pure ddH2O prior to transformation.
 +
**Arc time of 5.6 ms for both.
 +
*Transform M1-12(T7) and M1/2[1:2]-12(T7) into chemically competent BL21(DE3) cells.
 +
 
 +
==Liquid Culture==
 +
*Set up 2x 11 mL starter culture of M1/2[1:1]-12(T7)and M1/2[2:1]-12(T7) each.
 +
**One culture grown at 30 C, one grown at RT (~20 C)
 +
*Set up 11 uL of culture for M1-SeqAB2 (37 C).

Latest revision as of 05:37, 6 September 2015

iGEM UCLA




8/30/2015

Gel Purification

  • Used zymo kit to purify gels of M1-12 and M1/2[1:2]-12, elution in 12 uL
  • M1-12: 42.79 ng/uL
  • M1/2[1:2]-12: 57.8 ng/uL

Glycerol Stock Reconstitution

  • Plated M1/2[1:1]-12(T7), M1/2[2:1]-12(T7) and M1-SeqAB2 on LB + chlor plates.

RE digestion for M1-12, M1/2[1:2]-12

  • Digested M1-12, M1/2[1:2]-12 each with E,P and X,P.
  • Digest 5 uL of the samples purified today in a 50 uL reaction with 1 uL each of the respective enzymes.
  • Digest 37 C for 1.5 hrs, heat at 65 C for 20 min.
  • Digests were purified using Zymo clean and concentrator.

Ligation

  • For a 3:1 ligation to 50 ng of vector, 96 ng of 1324 bp insert is required
  • For a 5:1 ligation to 50 ng of vector, 160 ng of 1324 bp insert is required.
  • Ligated E,P digested products into pSB1C3.
  • Ligated X,P digested products into BBaK_525998.
  • For M1-12 ligations, used 10 uL of digested product to approximate a 3:1 insert:vector ratio due to insufficient amounts.
  • For M1/2[1:2]-12 ligations, used enough product to ligate at a 5:1 insert:vector ratio.
  • Ligate at 25 C for 1 hr, heat kill at 65 C for 20 min.

Transformation

  • Transform M1-12(1C3) and M1/2[1:2]-12(1C3) into DH%(alpha) electrocompetent cells.
    • Ligation products were dialyzed against ultra-pure ddH2O prior to transformation.
    • Arc time of 5.6 ms for both.
  • Transform M1-12(T7) and M1/2[1:2]-12(T7) into chemically competent BL21(DE3) cells.

Liquid Culture

  • Set up 2x 11 mL starter culture of M1/2[1:1]-12(T7)and M1/2[2:1]-12(T7) each.
    • One culture grown at 30 C, one grown at RT (~20 C)
  • Set up 11 uL of culture for M1-SeqAB2 (37 C).