Difference between revisions of "Team:UCLA/Notebook/Spider Silk Genetics/30 August 2015"
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− | =8/ | + | =8/30/2015= |
− | == | + | ==Gel Purification== |
− | *M1-12 | + | *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== |
− | *M1/2[1:1]-12( | + | *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. | |
− | *M1/2[1: | + | *Digest 5 uL of the samples purified today in a 50 uL reaction with 1 uL each of the respective enzymes. |
− | **1 | + | *Digest 37 C for 1.5 hrs, heat at 65 C for 20 min. |
− | ** | + | *Digests were purified using Zymo clean and concentrator. |
− | **3: | + | |
− | *M1/2[2:1 | + | ==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. |
− | *M1/2[ | + | *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). |
Latest revision as of 05:37, 6 September 2015
Contents
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).