SBB12Ntbk-Robert D O'Dowd

Robert D O'Dowd 2/16

sbb1214

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.

sbb1231

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

sbb1214

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.

sbb1231

Did not run any experiments

Robert D O'Dowd 2/21

sbb1231

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

sbb1214

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

sbb1231

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

sbb1214

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

sbb1231

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.

sbb1214

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

sbb1231

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

sbb1214

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

3/1 1231 Run gel on PCR products CI_TM and TM_PhoA and make sure bands show up Redo SOEPCR Lane 8 TMPhoA Lane 9 CITM Did not turn out well. Need to redo PCR

1214 One sample found 1214-1. Only 1 colony picked Miniprep procedure Label all 1213_1RO Final product 1213-1RO mPrep Need to run analytical gel

Robert D O'Dowd 3/2

3/2 1231 Need to start over. Reconstruct CITM and TM PhoA ca998/RDOSOECITM_R pBca9145jtk2768 500-1k Rack RdoSOETMPhoA_F/g00101 pBjh1601CK Bjh2128 1k-2k rack

1214 Should run another transformation in order to get more clones Need to run an analytical gel EcoRI XholI place in incubator 103-133

New transformation product Ligate 1214dig with pBca9525 Bca 1834 Nhe1 BamH1