Cloning the Ras Gene
Below you can see the primers designed to prepare
a PCR product containing the Ras gene
flanked by attB recombination
sites.
Forward attB
T
S
L Y K K A G
5'G
GGG ACA AGT TTG TAC AAA AAA GCA GGC TTC GAA
GGA GAT AGA ACC ATG
ACA GAA TAC AAG CTT GTG GTG G
3'
Reverse attB
5'GGG
GAC CAC TTT GTA CAA GAA AGC TGG GTC CTA
GTG
CTG CCG GAT CTC ACG CAC C 3'
c-H-Ras original sequence
1 ATG
ACA GAA TAC AAG CTT GTG GTG GTG GGC GCT GGA GGC GTG GGA AAG AGT
52 GCC CTG ACC ATC CAG CTG ATC CAG AAC CAT
TTT GTG GAC GAA TAC GAC CCC
102 ACT ATA GAG GAT TCC TAC CGG AAG CAG GTG GTC
ATT GAT GGG GAG ACG TGC
153 CTG TTG GAC ATC CTG GAT ACC GCC GGC CTG GAG
GAG TAC AGC GCC ATG CGG
204 GAC CAG TAC ATG CGC ACC GGG GAG GGC TTC CTG
TGT GTG TTT GCC ATC AAC
255 AAC ACC AAG TCT TTT GAG GAC ATC CAC CAG TAC
AGG GAG CAG ATC AAA CGG
306 GTG AAG GAC TCG GAT GAC GTG CCC ATG GTG CTG
GTG GGG AAC AAG TGT GAC
357 CTG GCT GCA CGC ACT GTG GAA TCT CGG CAG GCT
CAG GAC CTC GCC CGA AGC
408 TAC GGC ATC CCC TAC ATC GAG ACC TCG GCC AAG
ACC CGG CAG GGA GTG GAG
459 GAT GCC TTC TAC ACG TTG
GTG CGT GAG ATC CGG CAG CAC AAG CTG CGG AAG
510 CTG AAC CCT CCT GAT
GAG AGT GGC CCC GGC TGC ATG AGC TGC AAG TGT GTG
561 CTC TCC TGA
Nucleotides 499-570 were
not picked up by the PCR primers, instead we have inserted a stop codon.
Thus the ras gene cloned here is a truncated mutant, and does not contain
the amino acids 167-189.
You used these primers in a PCR reaction
and then ran the PCR products on a 1% agarose gel.
Click here to
see your results.