SBB12Ntbk-Robert D O'Dowd: Difference between revisions
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Zymo gel purification | Zymo gel purification | ||
label 1231ROTP, 1231ROCT, 1231MaybeTP | label 1231ROTP, 1231ROCT, 1231MaybeTP | ||
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[[Image:2012_02_21_gel1_ssb2012spring.jpg]]<br> | [[Image:2012_02_21_gel1_ssb2012spring.jpg]]<br> |
Revision as of 17:20, 12 April 2012
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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
sbb1231
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
sbb1214
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
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