SBB12Ntbk-Robert D O'Dowd

Robert D O'Dowd 2/16

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PCA on o16, o17, o12. Followed PCA protocol[1]

Created 100uM dilutions of o16 o17 o12.

Add nm weight x10= uL of ddH2O.

Label tube 1214. Place on PCA rack.

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PCR on ca998/rdoSOECI_TM-R and pBca 9145-jtk2768 Label CI TM 1231

PCR on rdoSOETM_PhoA-F/g00101 and pBjh 1601CK-Bjh2128 Label TM PhoA 1231

Followed PCR protocol [2]

Place CI TM 1231 on 500-1K bp rack

Place Label TM PhoA 1231 on 1K-2K bp rack

Robert D O'Dowd 2/17

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Zymo cleanup-small fragment cleanup. [3] Label 1214 PCA1

Amplification/phusion step. Template DNA added last. [4]

Template material and fragments for SOE placed in Eluded Products box.

Could not conduct SOEPCR gel or setup. Doing next tuesday.

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Did not run any experiments

Robert D O'Dowd 2/21

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Prep 6 uL product w/ 4 uL loading buffer Placed on wells 9, 10 labeled ROTP and ROCT Picture taken of gel->does not look correct Lane 9 has W shaped smear

I do not believe my recordings. Either I switched what lanes I believed was which or one of my products failed. I will switch assumptions and assum 9 ROCT and 10 ROTP Cut out band which correspond to correct size. Cut out extra band. 1K band maybe from lane 9 is ROCT Possible that 1K hidden in smear is actual TP product No 500 band appeared in lane 10 so its doubtful. Zymo gel purification label 1231ROTP, 1231ROCT, 1231MaybeTP

Lanes:

1	2	3	4	5	6	7	8	9	10
Au PCA2	TN sbb1213	DC A	DC B	2-log ladder	JS	MM	JW	RO TP	RO CT

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Cloning step. skip reassembly Named 1214 Cloning Followed digestion of wobble product protocol [5] except digested with NheI/BamHI Place in incubator at 1120 Store 1214 Cloning

Robert D O'Dowd 2/23

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SOE PCR Round 3 PCR 1231ROCT + 1231ROTP with ca998/g00101 Result labeled 1231PCR3 Placed in PCR rack for 1K-2K

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Taking digested product from tuesday->zymo column [6] Too much liquid for 1 column. Performing 2 rounds. Added 50 uL H2O. This amount was not correct should have only eluded w/8uL. Possible that product is too diluted for later steps to work. Ligate products Incubate on benchtop for 30 min 11:12-11:42 Waiting until Friday for transformation If there is an issue, it will likely be due to 6x dilution of vector Can restart at 1214Cloning

Robert D O'Dowd 2/24

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Run gel for PCR product Gel1 Lane 10 Labeled ROSOE3 1231 Place 1231 PCR3 back into box of products

Lanes:

1	2	3	4	5	6	7	8	9	10
MH PCRPDT 1218	MH PCRDIG 1218	empty	DC SB 1221	Ladder	empty	empty	empty	GF PCA	RO SOE3 1231

Cut out band at 2K place in tube labeled 1231SOE3 600uL ADB. Melt and place on ice 2 bands show up. one considerably brighter and heavier, Selected smaller band because it looks closer to the target 1800 bp I expect.

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Need to redo ligation Incubating on benchtop for 30 min 12:30-1:00 Label product 1213lig place on ice after 30 min mix 30 uL KCM Transfer 70 uL cell mixture to ligation tube. Wait 10 min 117-127 Probably dont have enough time to finish plating

Toss materials and retry tuesday

Robert D O'Dowd 2/28

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Complete zymo cleanup from product on Friday Label purified product 1231SOEPur Set up digestion from 1231SOEPur. Label 1231 dig. place at 37 at 1040-1140 Set up gel. 1202-1232 I expect to see a band at 1890

Lanes:

1	2	3	4	5	6	7	8	9	10
empty	AJ A+B pcrpdt	empty	RD 1231 dig	empty	2-log ladder	empty	empty	empty	empty

Dont see anything. Need to redo PCRs

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Redo ligation for sbb1214 Label 1214 Lig. 1028-1058 bench top incubate. Thaw cells and label cell prep. add 50 uL H2O. Transfer to new tube labeled 1214 Trans. Place on ice 1111-1121 Heatshok at 42C for 90 sec Place in shaker 1127-1227 Cutting incubatinon time by 15 min in order to complete during class period. 1212 Cells plated on Spec plates using alcohol sterilized wand. Plate labeled 2/28 RDO1214 pBca1825_1214 Plate given to Zach

Robert D O'Dowd 3/1

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Run gel on PCR products CI_TM and TM_PhoA and make sure bands show up Redo SOEPCR Run gel. Lane 8 TMPhoA Lane 9 CITM

Lanes:

1	2	3	4	5	6	7	8	9	10
Karin 1206	1224(1)	1224(2)	1224(3)	1203(1)	ladder	AJ	RO TM PhoI	RO CITM	Bad Load

Did not turn out well. Need to redo PCR

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One sample vial of cultured cells found 1214-1. Apparently only 1 colony was picked. Followed DNA Miniprep procedure. [7] Label all tubes used in process 1213_1RO Final product labeled 1213-1RO mPrep Need to run an analytical gel next time

Robert D O'Dowd 3/2

sbb1231 Because final expected product did not show up in gel last time, I need to start over. Reconstructing CITM and TM PhoA. PCR ca998/RDOSOECITM_R on pBca9145jtk2768. Mixture placed on 500-1k Rack PCR RdoSOETMPhoA_F/g00101 on pBjh1601CK Bjh2128. Mixture placed on 1k-2k rack

sbb1214 Since it is recommended to have 2 clones and I only 1 colony was picked, I should run another transformation in order to get more clones Digest pBca9525 Bca 1834 Nhe1 BamH1 and ligate with 1214dig Not enough time to transform and plate cells.

Running an analytical gel with EcoRI XholI. Digest placed in incubator 103-133

Lanes:

1	2	3	4	5	6	7	8	9	10
AJ A+B	AJ A+B	MM 1232-1	MM 1232-2	MM 1215-2	RO MP 1214	Ladder	MDP2	MDP1	MDL2

Robert D O'Dowd 3/6

sbb1231 Need to run gel to retrieve and purify SOEPCR products Lanes 1 (CITM) and 2 (TMPhoA) on Gel 3

Perform Zymo gel purification 1231 CITM2 and 1231 TMPhoA2 stored Solution labeled 1231 SOE3. Placed on 1-2K rack

sbb1214 Looking back on my notes, I did not run the second round of PCA w/ o16 and o12 on pca1. Restarting PCA1 with o12, o16, o17

Note from 3/15 My results from the analytical gel would seem to indicate that I had performed the correct reactions even though I did not record them in my notebook. I continue with the restart anyways.

Robert D O'Dowd 3/8

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1231 SOE3 digested with EcoRI and BamHI. Solution labeled 1231 Dig Placed in thermocycler 1007-1107

There is not enough time to ligate and plate.

Next week plan: Digest 1h -> Gel 30m -> Zymo 15m -> Ligate 30m -> Transform 1h -> Plate 15m

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Set up amplification for Pca1 mixture Label 1214Pca1 amp and place on Pca2 Rack for next stage Next week plan: Zymo -> Digestion -> Ligation -> Transform -> Plating Can run steps in parallel with 1231 at the ligation stage

Robert D O'Dowd 3/13

sbb1231 1231 SOE3 digested with EcoRI and BamHI to get 1231 Dig Placed in thermocycler from 1022-1122 Run gel. Lane 2 (RO 1231 dig) Save image Cut out the bright band in Lane 2 Add 600 mL ADB buffer and store for next time

sbb1214 1214Pca1 amp purified using zymo small fragment procedure. Labeled 1214 pur Begin digestion with NheI and BamHI. Result is 1214 dig. Placed in thermocycler 1022-1122 1214 dig run on gel. Lane 1 (RO 1214 dig) Add 600 mL ADB and store for next time

Lanes:

1	2	3	4	5	6	7	8	9	10
RO 1214 Dig	RO 1231 Dig	AJ A+B PCR digest	****	Lader	KA	KA	KA	KA	ladder

Robert D O'Dowd 3/15

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Zymo gel cleanup on RO 1231 Dig Run ligation reaction. pBca9525-Bca1834 digested with BamHI and EcoRI and ligated with RO 1231 Dig. Product is RO 1231 Lig Place on bench for 30 min 1031-1101 Add KCM to competent cells. Place on Ice for 10 min 1110-1120 Transform cells with RO 1231 Lig in thermomcycler at 42 C for 90 seconds. Place on ice for 1 min Place in incubator for 1 hour 1126-1226

Spilled my transformed cells and lost about half. This may cause contamination or not enough cells being plated

Plate on Spec plates and label RDO 1231 3/15

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Add 250 mL isopropanol. Proceed with Zymo small frag gel cleanup. Label resulting solution RO 1214 Dig Digest pBca9525-Bca1834 with NheI and BamHI. Ligate this with RO 1214 Dig. Result is RO 1214 Lig Incubate on bench for 30 in. 1031-1101. Add cells to ligation. Let it sit on ice 10 min 1110-1120 Place in thermocycler at 42 C for 90 seconds Place back on ice for 1 min Place in incubator for 1 hour. 1126-1226 Plate cells on Spec plates and label them RDO 1214 3/15

Robert D O'Dowd 3/20

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4 Colonies were picked by Zach. Turns out none of the cell cultures were viable and the plates I created were totally unusable. There are 2 large red clusters where cells survived. The colonies that were picked were likely contamination from when I leaked. It is also possible that the transformation totally failed.

Experiment stopped. sbb1231 inconclusive.

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4 colonies picked. All 4 cultures are viable. Performing miniprep on all 4. [8] Resulting solutions were labeled RO 1214 1, RO 1214 2, RO 1214 3, RO 1214 4 Running analytical digests on miniprepped plasmids

RO 1214 1 and 2 Digest with XbaI and NheI Expect bands at 3161 and 456 if successful and 3161 and 1174 if unsuccessful

RO 1214 3 and 4 Digest with BamHI and EcoRI Expect bands at 2472 and 1145 if successful and 2472 and 1863 if unsuccessful

Not enough time to incubate and run the gel. Performing the gel on Thursday.

Removing 1231ROCT, 1231PCR3, 1231CITM, 1214Pca1, 1231TMPhoA from storage in order to make space.

Robert D O'Dowd 3/22

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