Team:UCLA/Notebook/Honeybee Silk/17 July 2015

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Sequencing results

• One of the 5 sequencing reactions we sent out yesterday came back alright. There are two mutations, but luckily they are both silent :) You can see the sequencing allignment here and clicking on the alignments tab on the right side of the page.

Miniprep of 7/15 Transformation

I miniprepped the culture from 7/16 using the Zymo Miniprep Kit.

OD: 294.12 ng/uL

The sample was sent in for sequencing.

Transformation of BL21(DE3) with pET24a+silk

• Will using the miniprepped sample that came back with two silent mutations (pET+silk4)

1. Thaw cells (~80uL) on ice for 10 minutes.
2. Add 2uL of ligated product (diluted)
3. Place on ice for 5 minutes.
4. Rescue in 320 uL of SOC, incubate and shake for 1 hour.
5. Warm kanamycin plates at 37C.
6. Spin down bacteria to pellet, discard supernatant.
7. Add 101 uL of SOC, take 1uL and dilute 1:10.
8. Plate 100uL of each (1:1 and 1:10) on a plate.
9. Spread with beads, incubate at 37C (4 PM)

Double Digestion of honeybee silk in psb1c3 and BBa_K525998 and Cleanup

• I will be digesting our previous Biobrick (honeybee silk in psb1c3) and BBa_K525998 in preparation for ligation.
  • Honeybee silk+psb1c3: XbaI and PstI
  • BBa_K525998: SpeI and PstI

Component	Volume (out of 50uL)
NEB 2.1 Buffer	5uL
SpeI/XbaI	1uL
PstI	1uL
SIlk in psb1c3/BBa_K525998	4uL/3uL
Nuclease Free Water	39uL/40uL

Program

Step	Temperature	Time
Incubation	37C	1 hour
Heat Inactivation	80C	15 min
Hold	10C	Hold

I ran the digests on a 1% agarose gel against a 1kb ladder. The appropriate band sizes were present.

Ladder, Backbone, Insert

The backbone band and the 100bp band from the insert were gel extracted and purified using the Qiagen Gel Extraction Kit.

OD: Silk Insert: 4.45ng/uL Backbone: 30.89ng/uL

Ligation

• Using double digested samples of the Silk Insert and Backbone (3:1 molar ratio)

Component 3:1 insert:vector)	Volume (out of 20uL)
T4 Ligase Buffer	2 ul
BBa_K525998 backbone (30.89 ng/ul)	1 ul
Silk insert (4.45 ng/ul)	9uL
T4 Ligase	1 ul
Water	7 uL

• Incubated overnight at 16C