SBB12Ntbk-Robert D O'Dowd: Difference between revisions
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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 | |||
sbb1214 | sbb1214 | ||
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 | |||
==[[User:Robert D O'Dowd|Robert D O'Dowd]] 3/13== | ==[[User:Robert D O'Dowd|Robert D O'Dowd]] 3/13== | ||
sbb1231 | 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 | 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 | |||
[[Image:NEB_2-log_ladder.gif]][[Image:2012_03_13_gel3_ssb2012spring.jpg]]<br> | |||
Lanes:<br> | |||
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||RO 1214 Dig||RO 1231 Dig||AJ A+B PCR digest||****||Lader||KA||KA||KA||KA||ladder | |||
|} | |||
==[[User:Robert D O'Dowd|Robert D O'Dowd]] 3/15== | ==[[User:Robert D O'Dowd|Robert D O'Dowd]] 3/15== |
Revision as of 17:48, 12 April 2012
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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.
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
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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
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
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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
sbb1231
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
sbb1214
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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Robert D O'Dowd 3/20
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Robert D O'Dowd 3/22
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