IGEM:MIT/2007/Notebook/2007-7-9

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Nanodrop of Purified pSB3k3 from Mini-Prep of DB3.1 LC

1. 38.9 ng/µl
2. 38.7 ng/µl
3. 33.7 ng/µl
4. 33.8 ng/µl

Digestion of Purified pSB3k3 from this morning

Digestion 1:

25.6 µl pSB3k3 (38.9 ng/µl)
0.5 µl BSA
5.0 µl NEB3 Buffer
1.0 µl EcoRI
1.0 µl PstI
16.9 µl H2O
50µl Total

Digestion 2:

25.8 µl pSB3k3 (38.7 ng/µl)
0.5 µl BSA
5.0 µl NEB3 Buffer
1.0 µl EcoRI
1.0 µl PstI
16.7 µl H2O
50µl Total
  • Placed in 37C room at 4:30pm

Liquid Culture of DB3.1

Took two colonies from each plate (1A, 1B, 2A, 2B, 3A, 3B)

  • Placed in 37C room for overnight growth

Poured Plates

  • 16 LB+Amp (180µl in 450ml LB)
  • 28 LB+Kan (475µl in 475ml LB)

Purification of pSB3k3, F2620 and B0034 Digestion

pSB3k3: 3.5 ng/µl F2620: 17.7 ng/µl B0034: 19.9 ng/µl

  • Because B0034 is under 200bp, we had to use a different kit (Qiagen Nucleotide Removal Kit)
  • For pSB3k3 and F2620, we used the Qiagen PCR Purification Kit

Ligation of F2620 and B0034 into pSB3k3

[math]\displaystyle{ Mass_{insert}=4\frac{length_{insert}}{length_{backbone}}M_{backbone} }[/math]

Nanodrop of parts:

  • pSB3k3----3.5 ng/ul
  • B0034----19.9 ug/ul
  • F2620----17.7 ug/ul

Calculation

[math]\displaystyle{ \frac{50 ng}{3.5 ng/ \mu l}=14.3 \mu l }[/math] pSB3K3


[math]\displaystyle{ 17.7x = 4\frac{1070}{2750}50 }[/math]

[math]\displaystyle{ x=4.4\mu l }[/math]F2620

[math]\displaystyle{ 19.9 x = 4 \frac{17}{2750} 50 }[/math]

[math]\displaystyle{ x=.062 \mu l }[/math] B0034


Reaction Mix

'Note: In general the higher volume we make, the worse the transformation efficiency'

  • Used Rana's calculation, not the one above
  • 87.5 ng/ 25 ul -> Backbone
  • 12.5 ul -> 44 ng
  • 6 ul B0034 -> 100 ng
  • 6 ul F2020 -> 100 ng
  • 3 ul ligase buffer
  • .5 ul ligase
  • 2 ul H2O
  • ----
  • 30 ul (Ideally 20 ul or 10 ul) Room Temp, 90 min

Transformation

  • XL-1 Strain
  1. Aliquot 100 ul cells
  2. Add 1.7 ul B-mercaptoethanol
  3. Leave on ice for 10 min, swirling occasionally
  4. Add 2 ul DNA
  5. Ice for 30 min
  6. Heat pulse for 45 seconds at 42 degrees C (critical)
  7. Ice for 2 min
  8. Add 400 ul 42 degrees C heated LB
  9. 37 degree C room shaker 220 rpm for 1 hour
  • DH5alpha strain
  1. Aliquot 100 ul cells
  2. Add 2 ul DNA
  3. Ice for 30 min
  4. Heat pulse for 30 seconds
  5. Ice 2 min
  6. Add 1 ml 42 degrees C LB
  7. 37 degree room shaker 229 rpm for 1 hour

Plating

  1. Amp xL-1 20 ul
  2. Amp xL-1 200 ul
  3. Kan xL-1 20 ul
  4. Kan xL-1 200 ul
  5. Amp DH5alpha 100 ul
  6. Amp DH5alpha 200 ul
  7. Kan DH5alpha 100 ul
  8. Kan DH5alpha 200 ul

Transformation of Ligation Results into Rana's XL1-Blue Supercompetent Cells

Summary

  • Ligated together backbone parts
  • Transformed ligated backbone into 2 strains of competent cells
  • Plated competent cells

Expectations

The ligation of the backbone was tricky because of the length of the B0034 RBS. We may just need to PCR it on the end of the F2620 promoter.

The ligation was also tricky because we had very low concentrations of both our backbone and insert. The 3.5 ng/ul could be background.

Troubleshoot

  • Run backbone on gel
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