VIRAL SAFETY

ASDF

Sunday 1^st June

Protocol and Procedures

6/2

• Tested chemically competent cells through transformation

• Are cells contaminated?
• Are cells competent?

• The samples for transformation

#	Cells Tube label	DNA Tube label	Resistance before transformation	Resistance after transformation
1	K12 ER2738 5/20	p110+RBS (2) 4/23	Tet	Tet, Chlor
2	BW (-f) 5/30	p110+RBS (2) 4/23	Kan	Kan, Chlor
3	BW f-ep comp 5/22	p110+RBS (2) 4/23	Kan, Tet	Kan, Tet, Chlor
4	BW (+f) 5/30	p110+RBS (2) 4/23	Kan, Tet	Kan, Tet, Chlor
5	*BW23115 5/30	p110+RBS (2) 4/23	Kan	Kan, Chlor
2B	K12 ER2738	2B [from dis. kit]	Tet	Tet, Chlor
2P	BW f-ep comp 5/22	2P [from dis. kit]	Kan, Tet	Kan, Tet, Chlor

6/3

• Results from Transformation

Sample	Growth on Chlor	Growth on Kan	Growth on Kan+Tet	Growth on Amp
1 N	X	X	X	X
2 N	X	+	X	X
3 N	X	+	+	X
4 N	X	+	+	X
5 N	X	+	X	X
1	+	X	X
2	+	+	X
3	+	+	+
4	+	+	+
5	+	+	Many colonies but close to samp. 4
2P	+ (~100)	+	+
1- JW	+	X	X
2- JW	+	+	X
3- JW	+	+	+
4- JW	+	+	+
5-JW	+	+	X
2B- JW	+ (24)	X	X

• The transformations with DNA from the well (B2 and P2) had lower efficiencies than those with DNA from a mini-prep. Most likely this is due to drastic differences in DNA concentration (p110+RBS (2) 4/23 was at 254.4ng/ul)
• Conclusions

• None of the competent cells were contaminated
• All of the competent cells are in fact, competent

• Set up O/N of DH5-alpha cells to make competent tomorrow
• Planning ahead

• Wednesday

• Make chemically competent DH5alpha cells
• Test chemically competent cells
• Start phage DNA isolation protocol

• Thursday

• If transformation worked, transform cas9 into cells
• Finish phage DNA isolation protocol
• If primers come in, PCR on phage DNA (digestion method)

• Gibson?

• Friday

• Digest PCR

• Also pSB1C3 backbone

• Gel extraction
• Ligation

• Saturday

• Transformation

6/4

• Received cas9 from distribution kit (from GOLD): 4M
• Make chemically competent DH5alpha cells

• Labeled: 5a (that’s an alpha sign) 6/4 80 or 120ul

• Made chemically competent BW23115 with F-episome

• labeled: BWF+ 6/4
• 80ul per tube

• Transformation into new competent 5a (6/4)

• DNA

• p11+RBS(2) (4/23)                1ul
• 2B                                2ul
• 2P                                2ul
• 2P                                3ul
• No DNA

• Plated onto Chlor
• Also, thawed 5a cells for 45 minutes then returned to freezer

• Tomorrow, we will transform and test competency
• Also include non-competent control

• Isolation of single-stranded phagemid DNA using M13K07

• Added ER2738 colony to 50mL LB

• Plate was cold. Next time warm plate before pricking

• After 4 hours, OD was at 0.02. Waited 45 minutes and OD was at 0.08 (somehow). Therefore, we infected at OD 0.08
• Had started another culture when we did not think the first was growing. In incubator for about 1 hour. OD was 0.00. We infected anyway because last time it worked.
• Let infection proceed for 60 minutes then added 70ul of Kanamycin

• Primers came in!

• Gem001 F & R, Gem002 F & R, Gem003 F & R
• Resuspended DNA and diluted 1:10
• Other primers were ordered today

• Made assembly of plates with various antibiotic combinations

6/5

• Transformation results from 6/4

• No growth on negative (no DNA) control: no contamination
• > 400 colonies for positive (p11+RBS (2), diluted 1:10) control. Lawn when not diluted
• ~100 colonies for 2B (2ul) not diluted, 18 colonies on 1:10 dilution
• 3 colonies for 2P (2ul) not diluted, 0 on 1:10 dilution
• 20 colonies for 2P (3ul) not diluted, 1 on 1:10 dilution

• Made overnights of 2B and 2P to make freeze downs tomorrow
• Isolated single-stranded M13K07 DNA

• Final concentration = 5724 ng/ul

• Calculated from 1:10 dilution which had concentration of 572.4ng/ul

• For second sample in pair, we resuspended it in TE but did not proceed to DNA extraction steps
• For the second culture we started 6/4, we resuspended pellet in TBS and glycerol to preserve the M13 phage. Measured absorbances (before glycerol was added)

• #1

• 269 => 1.690A
• 320 => 0.103A

• #2

• 269 => 1.453
• 320 => 0.059

• To biobrick M13ori through biobrick assembly (the old-school way)

• PCR on Litmus 28i to amplify/biobrick M13ori

• Used primers Gem003 F & R
• Diluted Litmus 28i DNA 1:10

• Digestion of p11+RBS (1) to digest pSB1C3 bb with EcoRI-HF and PstI-HF
• Ran samples on gel and gel extracted pieces. We recieved very low yields (out of range for nano drop)

• M13 ori: 4.0 ng/ul
• pSB1C3: 1.8 ng/ul

• Digested M13 ori fragment despite poor extraction yield with EcoRI-HF and PstI-HF

• Used 1.5x as much DNA as instructed based on inaccurate concentration

• Ligation

• 10hr @ 16C, 10min @ 65C, 4ever @ 4C

• To biobrick M13 ori through Gibson Assembly (the cool-kids way)

• PCR on Litmus 28i

• Used primers Gem002 F & R
• Diluted Litmus 28i DNA 1:10

• PCR on pSB1C3 (p11+RBS (1))

• Used primers Gem001 F & R
• Diluted pSB1C3 DNA 1:3

6/6

• Ran gel of PCR products from (6/5)

• Recieved bands for pSB1C3 around 2000bp and M13ori around 500bp
• No contamination in pSB1C3 PCR negative control
• Band in M13ori negative control that is the same size as sample. Contaminated by sample?

• Gibson Assembly

• Diluted pSB1C3 and M13ori PCR products 1:10
• Incubated 60min @ 50C
• Also used provided pUC16 as positive control

• Transformation

• 1. p110+RBS                        Positive control
• 2. No DNA                        Negative control
• 3. Cas9 from distribution kit        so we can have more
• 4. Thaw and refreeze cells        Test competency of comp cells after thawed
• 5. Not chem comp cells        Negative control for the above
• 6. Ligation Product
• 7. Gibson product

• 7.2. Gibson product diluted 1:4

• 8. Gibson positive control

• 7.2. Gibson positive control diluted 1:4

• For the Gibson product and the positive control, we transformed 2ul of product and 2ul of 1:4 diluted product. NEB recomends the first if using their competent cells and the second if using cells from other companies. Our cells are from NEB but we made them competent ourselves so we tried both ways

• Plated on Chlor at concentrations of 170, 85, and 33 ug/mL

• Primers came in

• Resuspended and made 1:10 dilutions

6/7

• Results from 6/6 transformation

• 1. Positive control

• Lots of growth, ~300-400 on 1:10 dilution

• 2. No DNA negative control

• No growth

• 3. Cas9 from distribution kit

• 7 potential colonies (some are close to edges through) on non-diluted

• 4. Thawed then refroze cells

• Looks like (1)

• 5. Not chemically competent cells

• No growth

• 6. Ligation product

• 13 potential colonies (some are close to edge)

• 7. Gibson Assembly Product

• 170 -> No colonies
• 85 -> No colonies
• 33 -> 3 specks

• 7.2. Gibson Assembly product diluted 1:10

• 170 -> 1 speck
• 85 -> 3 colonies
• 33 -> 13 colonies

• 8. Gibson positive control

• No colonies

• 8.2. Gibson positive control diluted 1:4

• No colonies

• Made 6mL O/N cultures

• 4 from (3) cas9 plate
• 7 from (6) Ligation product
• 5 from (7.2 [85]) Diluted Gibson product on 85 ug/mL Chlor
• 8 from (7.2 [33]) Diluted Gibson product on 33 ug/mL Chlor

GOLD LAB

ASDF

Sunday 1^st June

Protocol and Procedures

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